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Publication numberUS5792852 A
Publication typeGrant
Application numberUS 07/977,696
Publication dateAug 11, 1998
Filing dateNov 16, 1992
Priority dateNov 16, 1992
Fee statusPaid
Publication number07977696, 977696, US 5792852 A, US 5792852A, US-A-5792852, US5792852 A, US5792852A
InventorsFernando J. R. do Couto, Roberto L. Ceriani, Jerry A. Peterson, Eduardo A. Padlan
Original AssigneeCancer Research Fund Of Contra Costa
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Polynucleotides encoding modified antibodies with human milk fat globule specificity
US 5792852 A
Abstract
A polynucleotide encodes a modified antibody, or single chains thereof. The modified antibody has a non-antigen-binding peptide such as the constant regions of an antibody of a first species, peptide hormones, enzymes, and peptide transmitters; and a binding peptide such as the unsubstituted light and heavy chains of the variable region of an antibody of a second species which binds the human milk fat globule (HMFG) antigen. The non-antigen-binding peptide is linked to at least one chain of the binding peptide, the chains may be linked to one another at a site other than the antigenic binding site, and at least one chain of the binding peptide has 1 to 46 amino acids substituted with amino acids selected from specific ones assigned to each site. The polynucleotide and other products are also provided in the form of compositions, with a carrier. The polynucleotides may be RNAs and DNAs, and are also provided as hybrid vectors carrying them, and as transfected cells expressing the modified antibodies or their single chains.
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Claims(63)
What is claimed as novel in Letters Patent of the United States is:
1. A polynucleotide comprising a nucleic acid encoding a modified antibody or single chains thereof which selectively binds the human milk fat globule (HMFG) antigen, and competes as well or better with the unmodified antibody for binding the HFMG, antigen comprising
a non-HMFG antigen-binding peptide from a first species; and
an HMFG antigen-binding peptide comprising the light and heavy chains of the variable region of an antibody of a second species; wherein
at least one chain of the non-HMFG antigen-binding peptide has 1 to 46 amino acids substituted with amino acids selected from the group consisting of the following amino acids at the specific sites and chains:
______________________________________    Light Chain     Heavy ChainPosition Amino Acid Substitution                    Amino Acid Substitution______________________________________1        D               PVLKARGHDE or Q2        WKCRHV or I     M or V3        V or L          LQR or K4        MPTQLV or I     M or L5        T               VD or E6        Q               QD or E7        TIAD or S       T or S8        PA or E         G or E9        LF or P         G10       T or S          DA or G11       NL or V         VF or L12       S or P          I or V13       V               KE or Q14       S or T          P15       PFI or L        G16       G               RSE or G17       TEQ or D        PA or S18       PS or Q         M19       V or A          R or K20       S               V or L21       I               S22       S               C23       C               TSE or A24                       V or A25                       S26                       G27                       F28                       AINS or T29                       F30                       S35       W36       IVHNYF or L     W37       WLVKRTHDE or Q  WIFLVMATG or Q38       Q               R39       R or K          Q40       FLKARTGQP or S  VA or S41       G               PLVARTSHNE or Q42       Q               G or E43       P or S          K44       P               SR or G45       EQR or K        L46       RV or L         Q or E47       WILMTSN or V    W48       FPLVMTS or I    STG or V49       Y or S          A57       WVTSGDNE or Q58       IYFLVMAT or Q59       S or P60       N or D61       T or R62       F63       IYPLKARSG or T64       D or G65       S66       G               R67       A or S          F68       VMCARSGQD or E  IS or T69       T               I70       D               L or S71       F               R72       T               VKRTSGHDE or N73       L               N or D74       NLRE or K       FPLVTGDNA or S75       L or I          EN or K76       IT or S         NTRK or S77       S or R          TNIVSM or R78       ALI or V        LA or V79       KGQ or E        FH or Y80       P or A          L81       ILVKMAGDN or E  E or Q82       D               M82a                      SDN or I82b                      IR or S82c                      PLVMAG or E83       MV or L         EKT or R84       LVARTS or G     SPVTI or A85       IM or V         WIYFPLMCRGD or E86       Y               D87       IMSHEFY or L    M or T88       C               A or G89                       IRVM or L90                       HF or Y91                       Y92                       C93                       ASHAV or T94                       TSDQLAWR or G98       F99       G100      IPVKRTGAS or Q101      G102      T103      IYMATGHDEQNR or K                    A or W104      LG or V         YH or G105      ILVTSGHNE or Q  THR or Q106      YLVKMRTD or I   G106a     PLVTI107      ILVMATSGNER or K                    AQ or T108                      STGM or L109                      IKLT or V100                      SL or T111                      V112                      T or S113                      PLVATG or S.______________________________________
2. The polynucleotide of claim 1, wherein the substitutions in the variable regions of the encoded modified antibody are selected from the group consisting of
______________________________________   Light Chain      Heavy ChainPosition   Amino Acid Substitution                    Amino Acid Substitution______________________________________ 1                       PVLKARGHDE or Q 2      WKCRH or I       M 3      V or L           LQR or K 4      PTQLV or I 5                       VD or E 6                       Q or D 7      IAD or S         T 8      A or E           E 9      F or P10      T                D or A11      N or V           V or F12      S                I13                       K or E14      S or T15      PFI or L16                       RS or E17      TEQ or D         P or A18      PS or Q19      V                R or K20                       V23                       TS or E24                       V28                       AINS or T36      IVHNYF or L37      WLVKRTHDE or Q   WIFLVMATG or Q39      R40      FLKARTGQP or S   VA or S41                       PLVARTSHNE or Q42                       G or E43      P44   S or R45      EQR or K46      R or V   Q47      WILMTSN or V48      FPLVMT or S      ST or G49      S57      WVTSGDNE or Q58      IYFLVMAT or Q59      S60      N61      T63      IYPLKARSG or T64      D67      A68      VMCARSGQD or E   I or S70                       L72                       VKRTSGHDE or N73                       N or D74      NLR or E         FPLVTGDN or A75      L                E or N76      I or T           NTRK or S77      S                TNIVSM or R78      AL or I          LA or V79      KG or Q          F or H80      P81      ILVKMAGDN or E   E 82a                     SDN or I 82b                     I or R 82c                     PLVMAG or E83      MV or L          EK or T84      LVARTS or G      SPVT or I85      IM or V          WIYFPLMCRG or D87      IMSHEFY or L     M88                       A or G89                       ITVM or L90                       H or F93                       ASHAV or T94                       TSDQLAWR or G100     IPVKRTGAS or Q103     IYMATGHDEQN or R A104     VL or G          Y or H105     ILVTSGHNE or Q   TH or R106     YLVKMRT or D106a    PLVT or I107     ILVMATSGNE or R  A or Q108                      STG or M109                      IKL or T110                      S or L112                      T113                      PLVATS or G.______________________________________
3. The polynucleotide of claim 2, wherein the substitutions in the variable regions of the encoded modified antibody are selected from the group consisting of
______________________________________   Light Chain      Heavy ChainPosition   Amino Acid Substitution                    Amino Acid Substitution______________________________________ 1                       PVLKARGHDE or Q 2      WKCR or H 3                       L 4      PT or Q36      IVH or N37      WLVKRTHD or E    WIFLVMATG or Q40      FLKARTG or Q41                       PLVARTSHNE or Q47      WILMTS or N48      FPLVMT or S57      WVTSGDNE or Q58      IYFLVMAT or Q63      IYPLKARS or G68      VMCARSG or Q72                       VKRTSGHD or E74                       FPLVTGD or N81      ILVKMAGDN or E 82c                     PLVMAG or E84      LVARTS or G85                       WIYFPLMCR or G87      IMSH or E94                       TSDQLA or W100     IPVKRT or G103     IYMATGHDE or Q104     L or G105     ILVTSGHNE or Q106     YLVKMRT or D106a    PLVTI107     ILVMATSGN or E109     I or K113     PLVAT or G.______________________________________
4. The polynucleotide of claim 2, wherein the substitutions in the variable regions of the encoded modified antibody are selected from the group consisting of
______________________________________   Light Chain      Heavy ChainPosition   Amino Acid Substitution                    Amino Acid Substitution______________________________________ 1                       Q 2      I                M 3      L or V           QR or K 5                       VD or E 6                       Q or D 7      IAD or S         T 8      A or E           E 9      F or P10      T                D or A11      N or V           V or F12      S                I13                       K or E14      T or S15      PFI or L16                       RS or E17      TEQ or D         P or A18      PS or Q19      V                R or K20                       V23                       TS or E24                       V28                       AINS or T36      YL or F37      Q39      R40      P or S           VA or S41                       T or S42                       G or E43      P44                       S or R45      EQR or K46      R or V           Q47      V48                       ST or G49      S59      S60      N61      T63      T64      D67      A68      GD or E          I or S70                       L72                       N73                       N or D74      NLR or E         A75      L                E or N76      I or T           NTRK or S77      S                TNIVSM or R78      AL or I          LA or V79      QG or Q          F or H80      P81                       E 82a                     SDN or I 82b                     I or R83      MV or L          EK or T84                       SPVT or I85      IM or V          D87      YL or F          M88                       A or G89                       ITVM or L90                       H or F93                       ASHAV or T94                       R or G100     AS or Q103     N or Q           A104     V                Y or H105                      TH or R106107     R                A or Q108                      STG or M109                      L or T110                      S or L112                      T113                      S or A.______________________________________
5. The polyribonucletide of claim 1, wherein the substitutions in variable regions of the encoded modified antibody are selected from the group consisting of
______________________________________   Light Chain      Heavy ChainPosition   Amino Acid Substitution                    Amino Acid Substitution______________________________________ 3                       Q or K 5                       V or E 7      S or T13                       Q or K14      T or S15      P or L17      E or D18      P or Q19                       R or K40      P or S           A or S42                       G or E44                       G or K45      Q or K68      G or E74      K or N           S or A77                       T or R81                       Q or E82                       N or I 82a                     S or N83      V or L84                       A or S88                       A or G89                       V or M110                      T or S113                      S or A.______________________________________
6. The polynucleotide of claim 1, wherein the non-HMFG antigen-binding peptide of the encoded modified antibody is selected from the group consisting of the constant regions of an antibody of the first species, hormones, enzymes, cytokines, and neurotransmitters.
7. The polynucleotide of claim 6, wherein the encoded constant regions are selected from the group consisting of complete human constant regions, and Fab, Fab', (Fab')2 fragments thereof and single chains thereof.
8. The polynucleotide of claim 5, wherein the non-HMFG antigen-binding peptide of the encoded modified antibody is selected from the group consisting of peptide hormones, enzymes, cytokines, and neurotransmitters.
9. The polynucleotide of claim 1, wherein the first and second antibody species of the encoded modified antibody are selected from the group consisting of murine, rat, goat, rabbit, canine, primate, bovine, ovine, equine, feline, pig, human, and guinea pig antibodies.
10. The polynucleotide of claim 1, wherein
the second species is non-human; and
the non-HMFG antigen-binding peptide is selected from the group consisting of complete human antibody constant regions, and Fab, Fab', (Fab')2 fragments thereof and single chains thereof.
11. The polynucleotide of claim 1, wherein the non-HMFG antigen-binding peptide is selected from the group consisting of human antibody constant regions which bind immunoglobulin, protein G, protein A and single chains thereof.
12. The polynucleotide of claim 1, wherein
the second species is non-human; and
the non-HMFG antigen-binding peptide is selected from the group consisting of human antibody constant regions which bind immunoglobulin, protein G, or protein A, and single chains thereof.
13. The polynucleotide of claim 1, wherein the modified antibody encoded by the nucleic acid is selected from the group consisted of polypeptides, comprising
the heavy chain constant region of an antibody of the first species linked to the heavy chain variable region of an antibody of the second species; and
the light chain constant region of an antibody of the first species linked to the light chain variable region of an antibody of the second species; wherein at least one of the variable region chains is substituted.
14. The polyribonucletide of claim 13, comprising the light and heavy chains of the variable regions of the encoded modified antibody, which are operatively linked to one another at a site other than the antigen binding site.
15. The polynucleotide of claim 9, wherein
the first species is murine; and
the second species is human.
16. The polynucleotide of claim 1, wherein the encoded modified antibody competes as well or better with the antibody expressed by the hybridoma cell ATCC No. HB 10028 for binding to the HMFG antigen.
17. The polynucleotide of claim 1, wherein the light chain variable region of the encoded modified antibody has 1 to 8 substitutions.
18. The polynucleotide of claim 1, wherein the light chain variable region is substituted at a position selected from the group consisting of positions 7, 14, 15, 17, 18, 40, 45, 68, 74, and 83.
19. The polynucleotide of claim 18, wherein the light chain variable region is substituted with S, T, P, E, P, P, G, and V at positions 7, 14, 15, 17, 18, 40, 68, and 83, respectively.
20. The polynucleotide of claim 1, wherein the heavy chain variable region of the encoded modified antibody has 1 to 12 substitutions.
21. The polynucleotide of claim 1, wherein the heavy chain variable region is substituted at a position selected from the group consisting of 3, 5, 13, 19, 40, 42, 44, 74, 77, 81, 82, 82a, 84, 88, 89, 110, and 113.
22. The polynucleotide of claim 21, wherein the heavy chain variable region is substituted with Q, V, R, G, T, N, A, and S at positions 3, 5, 19, 42, 77, 82, 88, and 113, respectively.
23. The polynucleotide of claim 1, wherein
the encoded HMFG antigen-binding peptide comprises a sequence selected from the group consisting of SEQ. ID No: 64 and SEQ. ID No: 65, and
the encoded non-HMFG antigen-binding peptide comprises a constant region peptide selected from the group consisting of the light and heavy chains of a human antibody constant region, each chain being operatively linked to one sequence at a site other than the HMFG antigen-binding site.
24. The polynucleotide of claim 1, wherein
the HMFG antigen-binding peptide comprises a sequence selected from the group consisting of SEQ. ID Nos: 67 through 73, each sequence being operatively linked to one another at a site other than the HMFG antigen-binding site, and SEQ. ID Nos: 75 through 81, each sequence being linked to one another at a site other than the HMFG antigen-binding site; and
the non-HMFG antigen-binding peptide comprises a constant region peptide selected from the group consisting of light and heavy chains of a human antibody constant region, each chain being operatively linked to one of the sequences at a site other than the HMFG antigen-binding site.
25. The polynucleotide of claim 1, wherein the nucleic acid encodes a polypeptide selected from the group consisting of
SEQ. ID No: 13 operatively linked to the heavy chain of a human antibody constant region at a site other than the HMFG antigen-binding site; and
SEQ. ID Nos: 67 to 73, each sequence being operatively linked to one another and to the light chain of a human antibody constant region at sites other than the HMFG antigen-binding site.
26. The polyribonucletide of claim 1, wherein the nucleic acid encodes a polypeptide selected from the group consisting of
SEQ. ID No: 11 linked to the light chain of a human antibody constant region at a site other than the HMFG antigen-binding site; and
SEQ. ID Nos: 75 to 81, each sequence being operatively linked to one another and to the heavy chain of a human antibody constant region at a site other than the HMFG antigen-binding site.
27. The polynucleotide of claim 1, wherein the nucleic acid encodes a polypeptide selected from the group consisting of
SEQ. ID Nos: 67 to 73, each sequence being operatively linked to one another and to the light chain of a human antibody constant region at a site other than the HMFG antigen-binding site; and
SEQ. ID Nos: 75 to 81, each sequence being operatively linked to one another and to the heavy chain of a human antibody constant region at a site other than the HMFG antigen-binding site.
28. The polynucleotide of claim 1, wherein the nucleic acid encodes the antibody expressed by the hybridoma cell ATCC No. HB 11200, or a single chain thereof.
29. The polynucleotide of claim 1, wherein the nucleic acid encodes a polypeptide selected from the group consisting of
the heavy chain constant region of an antibody of the first species operatively linked to the heavy chain variable region of an antibody of the second species at a site other than the HMFG antigen-binding site; and
the light chain constant region of an antibody of the first species operatively linked to the light chain variable region of an antibody of the second species at a site other than the HMFG antigen-binding site; wherein at least one of the chains of the variable region is substituted.
30. The polynucleotide of claim 29, wherein the heavy chain variable region has 1 to 46 substituted amino acids.
31. The polynucleotide of claim 29, wherein the light chain variable region has 1 to 46 substituted amino acids.
32. The polynucleotide of claim 29, wherein, in the encoded modified antibody
the first species is human; and
the second species is murine.
33. The polynucleotide of claim 1, wherein the nucleic acid encodes the antibody expressed by the hybridoma cell ATCC No. HB 11486, or a single chain thereof.
34. The polynucleotide of claim 1, wherein the nucleic acid encodes the antibody expressed by the hybridoma cell ATCC No. HB 11487, or a single chain thereof.
35. The polynucleotide of claim 1, comprising the light chain variable region of the encoded modified antibody expressed by the hybridoma cell line ATCC No. HB 10028 or a single chain thereof.
36. The polynucleotide of claim 1, comprising the heavy chain variable region of the encoded modified antibody expressed by the hybridoma cell line ATCC No. HB 1002 or a single chain thereof.
37. A composition of matter, comprising the polynucleotide of claim 1, and a non-lytic carrier.
38. The composition of claim 37, wherein the carrier is a pharmaceutically-acceptable carrier.
39. The polynucleotide of claim 1, wherein the encoded non-HMFG antigen-binding peptide comprises a chain selected from the group consisting of the light and heavy chains of the constant region of an antibody of the first species.
40. The polynucleotide of claim 1, wherein the nucleic acid encodes a fragment selected from the group consisting of Fab, Fab', (Fab')2 fragments and single chains of the antibody expressed by the hybridoma cell ATCC No. HB 11200.
41. The polynucleotide of claim 1, wherein the nucleic acid encodes a fragment selected from the group consisting of Fab, Fab', (Fab')2 fragments and single chains of the antibody expressed by the hybridoma cell ATCC No. HB 11486.
42. The polynucleotide of claim 1, wherein the nucleic acid encodes a fragment selected from the group consisting of Fab, Fab', (Fab')2 fragments and single chains of the antibody expressed by the hybridoma cell ATCC No. HB 11487.
43. The polynucleotide of claim 34, comprising the heavy chain variable region of the encoded modified antibody or a single chain thereof.
44. The polynucleotide of claim 33, comprising the heavy chain variable region of the encoded modified antibody or a single chain thereof.
45. The polynucleotide of claim 34, comprising the light chain variable region of the encoded modified antibody or a single chain thereof.
46. The polynucleotide of claim 33, comprising the light chain variable region of the encoded modified antibody or a single chain thereof.
47. A labeled polynucleotide, comprising the polynucleotide of claim 1; and a label operatively linked thereto.
48. A radiolabeled polynucleotide, comprising the labeled polynucleotide of claim 47, wherein the label comprises a radiolabel.
49. The polynucleotide of claim 1, which is a DNA.
50. The polynucleotide of claim 1, which is an RNA.
51. A vector, having the polynucleotide of claim 1 operatively linked thereto.
52. The vector of claim 51, wherein the vector has the polynucleotide operatively linked in reading frame.
53. The vector of claim 51, comprising an expression vector.
54. A composition, comprising the vector of claim 51, and a carrier.
55. A host cell, transfected with the polynucleotide of claim 1.
56. The host cell of claim 55, wherein the polynucleotide is operatively linked to a vector.
57. The transfected host cell of claim 55, which is the hybridoma cell ATCC HB 11200.
58. The transfected host cell of claim 55, which is the hybridoma cell ATCC HB 11486.
59. The transfected host cell of claim 55, which is the hybridoma cell ATCC HB 11487.
60. A composition, comprising the transfected host cell of claim 55, and a medium or carrier.
61. The polynucleotide of claim 28, comprising the heavy chain variable region of the encoded modified antibody.
62. The polynucleotide of claim 28, comprising the light chain variable region of the encoded modified antibody.
63. The polynucleotide of claim 1, comprising a chain selected from the group consisting of the light and heavy chains of the variable region expressed by the hybridoma cell line ATCC No. HB 10028.
Description
BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the diagnosis, immunization, and therapy of neoplastic tumors, particularly carcinomas by means of specifically targeted analogue peptides comprising amino acid sequences encompassing, for instance, the complementarity determining regions (CDRs), and analogues of the variable (Fv) region of anti-carcinoma antibodies, among others. The carcinoma specific peptides are provided as a single amino acid chain having the specificity of the non-human antibody Fv regions of the light or heavy chains, or as paired chains, either by themselves or bound to other polymers such as synthetic polymers or oligopeptides resulting in chimeric antibodies, and more particularly analogues of human/non-human chimeric antibodies or other polymeric constructs. The analogue peptides comprise sequences derived from variable regions of heterogeneous antibodies specific for human carcinoma antigens that elicit a lesser immunological response in humans than whole heterologous non-human antibodies. The peptides of the invention are useful for in vivo and in vitro diagnosis and therapy of carcinoma. Anti-idiotype polypeptide and analogues thereof are suitable for immunizing humans or other animals against carcinoma. Polynucleotide sequences, hybrid vectors and host cells encoding the analogue peptide and anti-idiotype polypeptides, hybrid vectors and transfected hosts are useful for preparing the peptides disclosed herein.

2. Description of the Background

Carcinomas result from the carcinogenic transformation of cells of different epithelia. Two of the most damaging characteristics of carcinomas are their uncontrolled growth and their ability to create metastases in distant sites of the host, particularly a human host. It is usually these distant metastases that cause serious consequences to the host, since frequently the primary carcinoma may be, in most cases, removed by surgery. The treatment of metastatic carcinomas, that are seldom removable, depends on irradiation therapy and systemic therapies of different natures. The systemic therapies currently include, but not fully comprise, chemotherapy, different immunity-boosting medicines and procedures, hyperthermia and systemic monoclonal antibody treatment. The latter can be labeled with radioactive elements, immunotoxins and chemotherapeutic drugs.

Radioactively labeled monoclonal antibodies were initially used with success in lymphomas and leukemia, and recently in some carcinomas. The concept underlying the use of labeled antibodies is that the labeled antibody will specifically seek and bind to the carcinoma and, the radioactive element, through its decay, will irradiate the tumor in situ. Since radioactive rays travel some distance in tumors it is not necessary that every carcinoma cell bind the labeled antibody. The specificity of the monoclonal antibodies will permit a selective treatment of the tumor while avoiding the irradiation of innocent by-stander normal tissues, that could be dose limiting. Chemotherapy produces serious toxic effects on normal tissues, making the chemotherapy of carcinomas less than desirable, and the use of radiolabeled monoclonal antibodies a valid alternative.

Non-human antibodies raised against human epitopes have been used for the diagnosis and therapy of carcinomas as is known in the art. Also known are the methods for preparing both polyclonal and monoclonal antibodies. Examples of the latter are BrE-2, BrE-3 and KC-4 (e.g., U.S. Pat. Nos. 5,077,220; 5,075,219 and 4,708,930.

The KC-4 murine monoclonal antibody is specific to a unique antigenic determinant, the "antigen", and selectively binds strongly to neoplastic carcinoma cells and not to normal human tissue (U.S. Pat. No. 4,708,930 to Coulter). The antigen appears in two forms in carcinoma cells, only the smaller of these forms being expressed in the cell membrane. The larger form appears only in the cytoplasm and has an approximate 490 Kdalton molecular weight (range of 480,000-510,000). The second form occurs at a higher density of expression, is found both in the cytoplasm and the membrane of carcinoma cells and has an approximate 438 Kdalton molecular weight (range of 390,000-450,000) as determined by gel electrophoresis with marker proteins of known molecular weights. Labeled KC-4 was applied to the diagnosis and medical treatment of various carcinomas, particularly adenocarcinoma and squamous cell carcinoma regardless of the human organ site of origin.

The BrE-3 antibody (Peterson et al., Hybridoma 9:221 (1990); U.S. Pat. No. 5,075,219) was shown to bind to the tandem repeat of the polypeptide core of human breast epithelial mucin. When the mucin is deglycosylated, the presence of more tandem repeat epitopes is exposed and the binding of the antibody increases. Thus, antibodies such as BrE-3 bind preferentially to neoplastic carcinoma tumors because these express an unglycosylated form of the breast epithelial mucin that is not expressed in normal epithelial tissue. This preferential binding combined with an observed low concentration of epitope for these antibodies in the circulation of carcinoma patients, such as breast cancer patients, makes antibodies having specificity for a mucin epitope highly effective for carcinoma radioimmunotherapy. A 90 Y-BrE-3 radioimmunoconjugate proved highly effective against human breast carcinomas transplanted into nude mice. Human clinical studies showed the 90 Y-BrE-3 radioimmunoconjugate to considerably reduce the size of breast tumor metastases without any immediate toxic side effects. Moreover, an 111 In-BrE-3 radioimmunoconjugate was successfully used for imaging 15 breast cancer patients, providing excellent tumor targeting in 13 out of 15 of the patients. Out of all the breast tumor metastases occurring in another study, 86% were detected by 111 In-BrE-3. Unfortunately, 2 to 3 weeks after treatment, the patients developed a strong human anti-mouse antibody (HAMA) response that prevented further administration of the radioimmunoconjugate. The HAMA response, which is observed for numerous murine monoclonal antibodies, precludes any long-term administration of murine antibodies to human patients. Similarly, other heterologous antibodies, when administered to humans, elicited similar antibody responses. The anti-heterologous human response is, thus, a substantial limiting factor hindering the successful use of heterologous monoclonal antibodies as therapeutic agents, which could, otherwise, specifically annihilate breast carcinomas, causing little or no damage to normal tissue and having no other toxic effects.

Chimeric antibodies are direct fusions between variable domains of one species and constant domains of another. Mouse/human chimeric antibodies prepared from other types of B cells binding to other types of antigenic determinants have been shown to be less immunogenic in humans than whole mouse antibodies. These proved to be less immunogenic but still in some cases there is a mounted immune response to the rodent variable region framework region (FR). A further reduction of the "foreign" nature of the chimeric antibodies was achieved by grafting only the CDRs from a rodent monoclonal into a human supporting framework prior to its subsequent fusion with an appropriate constant domain, (European Patent Application, Publication No. 239,400 to Winter; Riechmann, et al., Nature 332:323-327 (1988). However, the procedures employed to accomplish CDR-grafting often result in imperfectly "humanized" antibodies. That is to say, the resultant antibody loses avidity (usually 2-3 fold, at best).

The ligand binding characteristics of an antibody combining site are determined primarily by the structure and relative disposition of the CDRs, although some neighboring residues also have been found to be involved in antigen binding (Davies, et al., Ann. Rev. Biochem. 59:439-473 (1990)).

The technologies of molecular biology have further expanded the utility of many antibodies by allowing for the creation of class switched molecules whose functionality has been improved by the acquisition or loss of complement fixation. The size of the bioactive molecule may also be reduced so as to increase the tissue target availability of the antibody by either changing the class from an IgM to an IgG, or by removing most of the heavy and light chain constant regions to form an Fv antibody. Common to all of these potentially therapeutic forms of antibody are the required complementary determining regions (CDRs), which guide the molecule to its ligand, and the framework residues (FRs) which support the CDRs and dictate their disposition relative to one another. The crystallographic analysis of numerous antibody structures revealed that the antigen combining site is composed almost entirely of the CDR residues arranged in a limited number of loop motifs. The necessity of the CDRs to form these structures, combined with the appreciated hypervariability of their primary sequence, leads to a great diversity in the antigen combining site, but one which has a finite number of possibilities. Thus, its hypermutability and the limited primary sequence repertoire for each CDR would suggest that the CDRs derived for a given antigen from one species of animal would be the same derived from another species. Hence, they should be poorly immunogenic, if at all, when presented to a recipient organism.

Accordingly, there is still need for a product of high affinity and/or specificity for carcinoma antigens suitable for the detection and therapy of carcinomas which elicits a lesser antibody response than whole non-human antibodies orchimeric antibodies containing, for instance the entire non-human variable region.

SUMMARY OF THE INVENTION

This invention relates to an analogue peptide or a glycosylated derivative which specifically binds to an antigen found on the surface or in the cytoplasm of carcinoma cells or released by the cells, the analogue peptide consisting essentially of at least one CDR or variable region of the light or heavy chains of an antibody of a first species having affinity and specificity for an antigen found on the surface or the cytoplasm of a carcinoma cell or released by the cells, wherein preferably about 1 to at least 46 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of a second species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of to about 1 to at least 10 amino acids, combinations thereof, combinations thereof with variable regions or analogues thereof, wherein each analogue peptide is operatively linked to at least one other peptide or analogue thereof, or mixtures thereof. The analogue is also provided as a fusion protein, their corresponding DNAs hybrid vectors, transfected hosts and RNAS. This invention also encompasses a method of producing an analogue peptide or hybrid analogue peptide by recombinant technology. Also provided herein are in vivo an in vitro methods of diagnosing and for the therapy of a carcinoma.

Also disclosed herein is an anti-idiotype polypeptide, comprising polyclonal antibodies raised against the analogue peptide of this invention, monoclonal antibodies thereof, fragments thereof selected from the group consisting of Fab, Fab', (Fab')2, CDRs, variable regions and analogues thereof described above, an anti-carcinoma vaccine, a vaccination kit, a method of vaccinating against carcinoma, and a method of lowering the serum concentration of a circulating antibody with the anti-idiotype polypeptide of this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention arose from a desire by the inventors to improve on antibody technology suitable for use in diagnostic, vaccine and therapeutic applications. The monoclonal antibodies obtained up to the present time have been prepared by fusing immortalized cell lines with B-cells of non-human origin such as murine, rat, rabbit, goat, and the like. Many of these hybridomas can produce monoclonal antibodies that have desirable binding properties such as high affinity and/or specificity for human carcinoma antigens, and are also produced in large amounts. However, in general, non-human antibodies may only be administered once to humans due to the detrimental effects they produce. This is true for most xenogeneic antibodies being administered to a mammalian animal. For example, the repeated administration of mouse antibodies to a human subject elicits a strong human anti-mouse antibody (HAMA) response, which precludes their further utilization as therapeutic agents in humans. These non-human antibodies initiate an immediate adverse reaction in many patients and are, thus, rendered ineffective as therapeutic agents. Non-human-human chimeric antibodies and non-human CDR "grafted"-human antibodies may have low affinity and/or specificity for their antigens. On the other hand, human monoclonal hybridoma cell lines have not been very stable and have, therefore, not been suitable for the large scale, repeated production of monoclonal antibodies.

The present inventors, thus, have undertaken the preparation of anti-carcinoma human and non-human CDRs and non-human variable regions of antibodies, having affinity and specificity for an antigen found on the surface or the cytoplasm of a human carcinoma cell or released by the cells, wherein about 1 to 46 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in human antibodies, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least up to 10 amino acids, alone or with an N-terminal fragment of about 1 to at least up to 10 amino acids, to lower or even circumvent the anti-xenogeneic response. To preserve substantial binding specificity the present invention utilizes CDRs and/or analogues of varying lengths of the variable regions of light and/or heavy chains of mouse, rat, rabbit, goat, horse, primate such as human and simian, bovine, and guinea pig antibodies, among others.

The present inventors have found, surprisingly, that these analogue antibody fragments substantially preserve the binding and specificity characteristics of the whole antibody while eliciting a lesser detrimental immunological. However, the simple preservation of the binding region of an antibody does not ensure that the binding characteristics of the antibody will be maintained. Antibodies are glycopolypeptides that are folded into specific conformations. When the glycoside portion of the molecule or portions of the amino acid sequence are perturbed or excised, the folding pattern of the molecule is generally perturbed. Thus, any deletion or modification of sequences of an antibody must be made taking into consideration that its folding-dependent properties may be diminished or even obliterated if the folding is affected, even though the amino acid sequences involved in the binding of the antigen are preserved.

The present inventors have selected the following strategy for the preparation and manufacture of the analogue peptides and hybrid peptides of this invention. The cDNAs that encode the variable chains of an antibody may be obtained by isolation of mRNA from a hybridoma cell and reverse transcription of the mRNA, amplification of the cDNA by PCR and insertion of the DNA into a vector for sequencing and restriction enzyme cutting. The cDNAs encoding the CDR or Fv region fragments of the light (VL) and heavy (VH) chains of an antibody having affinity and specificity for a carcinoma cell antigen may be reverse transcribed from isolated mRNA. The variable region cDNAs may then be modified with predesigned primers used to PCR amplify them, cloned, into a vector optionally carrying DNA sequences encoding, e.g., a constant region(s), optionally sequenced, and then transfected into a host cell for expression of the analogue gene products. The binding specificity characteristics of the analogue peptides may be then determined and compared to those of the whole antibodies.

X-ray crystalographic studies have repeatedly demonstrated that the framework structures of the FV s of different antibodies assume a canonical structure regardless of the species of origin, amino acid sequence, or ligand specificity. This is generally taken as evidence that the ligand-binding characteristics of an antibody combining site are determined primarily by the structure and relative disposition of the CDRs, although some neighboring framework residues also have been found to be involved in antigen-binding. Thus, if the fine specificity of an antibody is to be preserved, its CDR structures, and probably some of the neighboring residues, their interaction with each other and with the rest of the variable domains, must also be maintained. These crystallographic studies point to the possible need for retaining most, if not all, of the many interior and inter-domain contact residues since the structural effects of replacing only a few of them cannot be predicted.

While at first the necessity of keeping these amino acids might seem to defeat the humanization goal of decreasing immunogenicity, the actual number of amino acids that must be retained is usually small because of the striking similarity between human and murine variable regions. Moreover, many, if not most, of the retained amino acids possess side chains that are not exposed on the surface of the molecule and, therefore, may not contribute to the antigenicity.

The challenge in humanizing the variable regions of a non-human antibody, e.g., a murine antibody, thus begins with the identification of the "important" xenogenenic amino acids. "Important" amino acids are those that are involved in antigen binding, contact the CDRs and the opposite chains, and have buried side-chains. Ideally, these residues would be readily identified from a well characterized three-dimensional structure. When direct structural data are not available, it is, fortunately, still possible to predict the location of these important amino acids from the knowledge of other antibody structures, especially those whose variable light and heavy regions belong to the same class. The classes of variable regions can be determined from their amino acid sequence.

A method by which these important amino acids are identified has been described for the case of the amino acids with buried side chains by Padlan, E. A. (Padlan, E. A., "A Possible Procedure for Reducing the Immunogenicity of Antibody Variable Domains While Preserving Their Ligand-Binding Binding Properties", Molecular lmmmunology, 28:489-4948 (1991)). Various antibody variable region structures were compared using a computer program that determines the solvent accessibility of the framework residues as well as their contacts with the opposite domain (Padlan, E. A. (1991), supra). Surprisingly, a close examination of the fractional solvent accessibility reveals a very close similarity in the exposure patterns of the VH and the VL domains. Put in simple terms, this means that regardless of the particular antibody in question, and of its amino acid sequence, the buried residues occupy the same relative positions in most antibodies.

A similar analysis can be done by computer modeling, to determine which amino acids contact the CDRs and which contact the opposite domain. At this point, the Fab structures that are currently in the Protein Data bank (Bernstein, F. C., et al., J. Mo. Biol. 112:535-542 (1977)) may be examined to determine which FRs are probably important in maintaining the structure of the combining site. Thus, after a close inspection of many high resolution three-dimensional structures of variable regions, the positions of all important framework amino acids, that is, those that contact the CDRS, the opposite domain, and those whose side chains are inwardly pointed, may be tabulated. Keeping these amino acids, as well as those from the CDRs, and finally those FR amino acids that may be involved in ligand binding, should insure to a great extent the preservation of affinity. The precise identification of FR amino acids that are involved in ligand-binding cannot be generalized since it varies for different antibodies. Nevertheless, conservative decisions can be made to preserve the amino acids located in FR that have a high probability of contacting the antigen. These regions are located immediately adjacent to the CDRs and at the N-terminus of both chains, because the surfaces of these regions are contiguous with the CDR surfaces.

Surprisingly, it is possible to keep all of these important amino acids in a heterologous humanized antibody and still increase dramatically the similarity with a human consensus sequence. That is, the final number of amino acids with mouse identities differing from human identities that are kept is typically small. This is usually possible because human frameworks that are similar to the mouse frameworks, especially at the positions of the important amino acids, can be found. This is because many of the important amino acids have the same identities in both mouse and human antibodies.

All the amino acids that are determined to be not important by the method described above may be completely replaced by their corresponding human counterparts. The surface of the finally humanized antibody should look very much like that of a human antibody except for the antigen binding surfaces. The original shape of those binding surfaces, however, is maintained by leaving the internal composition of the antibody intact, preserving inter-domain contacts and by keeping very few key amino acids that contact the CDRs.

a) Choosing the Best Human Framework to Use in the "Humanization" of an Antibody When Its Structure Is Known

At the present time, there are 11 Fab structures for which the atomic coordinates are known and have been placed in the Protein Data Bank as shown in Table 1 below, 2 from human and 9 from murine antibodies.

              TABLE 1______________________________________Fab Structures for Which Coordinatesare in the Protein Data BankANTIBODY     RESOLUTION (A)                     R-VALUE  PDB CODE______________________________________HUMAN: NEWM      2.0          0.46   3FAB  KOL       1.9          0.189  2FB4MURINE:  McPC603   2.7          0.225  1MCP  J539      1.95         0.194  2FBJ  HyHEL-5   2.54         0.245  2HFL  HyHEL-10  3.0          0.24   3HFM  R19.9     2.8          0.30   1F19  4-4-20    2.7          0.215  4FAB  36-71     1.85         0.248  6FAB  B13I2     2.8          0.197  1IGF  D1.3      2.5          0.184  1FDL______________________________________

The contacts between side chains in the variable domains of the 11 Fabs have been collected and are presented in Tables 2 to 4 below. The FR in the VL domains that contact CDRs are listed in Table 2 below.

                                  TABLE 2__________________________________________________________________________VL Framework Residues That Contact CDR Residuesin Fabs of Known Three-Dimensional StructureANTIBODYPOSITION J539 McPC603           HyHEL-10                 HyHEL-5                      R19.9                           4-4-20                                36-71                                     B13I2                                          D1.3 NEWM KOL__________________________________________________________________________ 1    GLU(2)      ASP(5)           ASP(10)                 ASP(3)    ASP(8)                                ASP(4)    ASP(11) 2    ILE(11)      ILE(15)           ILE(17)                 ILE(13)                      ILE(5)                           VAL(9)                                ILE(20)                                     VAL(9)                                          ILE(10)                                               SER(3) 3         VAL(3)           VAL(2)                 VAL(3)                      GLN(2)                           VAL(2)                                GLN(2)                                     LEU(6)    VAL(2) 4    LEU(7)      MET(6)           LEU(6)                 LEU(10)                      MET(9)                           MET(13)                                MET(7)                                     MET(6)                                          MET(7)                                               LEU(4)                                                    LEU(6) 5         THR(1)          THR(1)                           THR(2)              THR(1) 7                                        THR(4)22                                        SER(6)23    CYS(1)      CYS(1)           CYS(2)                 CYS(2)                      CYS(1)                           CYS(1)                                CYS(1)              CYS(1)35    TRP(3)      TRP(2)           TRP(4)     TRP(2)    TRP(6)                                     TRP(4)                                          TRP(4)                                               TRP(1)                                                    TRP(2)36    TYR(12)      TYR(16)           TYR(8)                 TYR(10)                      TYR(22)                           TYR(13)                                TYR(15)                                     TYR(8)                                          TYR(14)                                               TYR(13)                                                    TYR(11)45                         LYS(12)                           LYS(5)46    PRO(3)      LEU(6)           LEU(4)                 ARG(15)                      LEU(5)                           VAL(14)                                LEU(5)                                     LEU(10)                                          LEU(6)                                               LEU(2)                                                    LEU(6)48    ILE(1)      ILE(1)           ILE(1)               ILE(3)                                     ILE(2)                                          VAL(1)    ILE(1)49    TYR(28)      TYR(29)           LYS(13)                 TYR(12)                      TYR(40)                           TYR(22)                                TYR(22)                                     TYR(16)                                          TYR(25)   TYR(25)58    VAL(3)      VAL(3)           ILE(1)                 VAL(6)                      VAL(6)                           VAL(5)                                VAL(4)                                     VAL(5)                                          VAL(1)    VAL(6)60         ASP(1)               ASP(2)    ASP(4)         ASP(2)62                    PHE(1)    PHE(1)                                PHE(1)66                                                  LYS(2)                                                    LYS(11)67         SER(3)                              SER(1)69         THR(3)           THR(3)          THR(5)                                THR(1)                                     THR(4)                                          THR(1)                                               SER(1)70         ASP(2)          ASP(1)    ASP(6)         SER(2)71    TYR(14)      PHE(23)           PHE(17)                 TYR(17)                      TYR(24)                           PHE(17)                                TYR(17)                                     PHE(19)                                          TYR(16)                                               ALA(3)                                                    ALA(4)88    CYS(1)    CYS(2)     CYS(1)                           CYS(1)                                CYS(1)                                     CYS(1)                                          CYS(2)    CYS(1)98    PHE(8)      PHE(8)           PHE(10)                 PHE(5)                      PHE(8)                           PHE(4)                                PHE(8)                                     PHE(14)                                          PHE(14)                                               PHE(3)                                                    PHE(7)__________________________________________________________________________

Those FR in the VH domains that contact CDRs are listed in Table 3 below.

                                  TABLE 3__________________________________________________________________________VH Framework Residues That Contact CDR Residuesin Fabs of Known Three-Dimensional StructureANTIBODYPOSITION J539 McPC603           HyHEL-10                 HyHEL-5                      R19.9                           4-4-20                                36-71                                     B13I2                                          D1.3 NEWM KOL__________________________________________________________________________ 1                                   GLU(3) 2    VAL(11)      VAL(3)           VAL(8)     VAL(1)    VAL(7)                                     VAL(3)                                          VAL(12)   VAL(9) 4    LEU(2)      LEU(5)           LEU(5)     LEU(2)                           LEU(1)                                LEU(1)                                     LEU(1)                                          LEU(1)    LEU(1)24         THR(2)           VAL(6)          ALA(1)27    PHE(3)      PHE(2)     TYR(14)                      TYR(11)                           PHE(26)                                TYR(4)                                     PHE(4)                                          PHE(4)                                               THR(1)                                                    PHE(3)28    ASP(9)      THR(5)     THR(3)                      THR(6)                           THR(4)                                THR(2)                                     THR(3)    SER(1)                                                    ILE(2)29    PHE(4)      PHE(4)     PHE(10)                      PHE(7)                           PHE(13)                                PHE(6)                                     PHE(3)                                          LEU(1)    PHE(4)30              THR(2)     THR(6)                           SER(7)              ASP(6)36                                   TRP(2)37         VAL(1)          VAL(1)              VAL(1)                                               VAL(2)                                                    VAL(1)38    ARG(1)      ARG(2)           ARG(4)                 LYS(2)                      LYS(1)                           ARG(4)                                LYS(2)                                     ARG(1)         ARG(3)40                    ARG(1)46    GLU(3)      GLU(4)           GLU(1)                 GLU(27)                      GLU(3)                           GLU(4)                                GLU(9)    GLU(1)    GLU(1)47    TRP(21)      TRP(29)           TYR(20                 TRP(21)                      TRP(13)                           TRP(18)                                TRP(21)                                     TRP(23)                                          TRP(19)                                               TRP(22)                                                    TRP(15)48    ILE(1)      ILE(1)           MET(6)                 ILE(12)                      ILE(13)                           VAL(1)                                ILE(9)                                     VAL(3)                                          LEU(1)                                               ILE(2)                                                    VAL(1)49         ALA(2)               ALA(2)    AL(2)          ALA(2)66              ARG(11)         ARG(3)    ARG(2)    ARG(2)                                                    ARG(1)67    PHE(4)      PHE(10)           ILE(9)                 ALA(1)    PHE(11)                                THR(5)                                     PHE(12)                                          LEU(6)                                               VAL(2)                                                    PHE(10)68         ILE(1)          THR(1)                           THR(11)                  THR(2)69    ILE(8)      VAL(6)           ILE(8)                 PHE(12)                      LEU(5)                           ILE(20)                                LEU(6)                                     ILE(11)                                          ILE(8)                                               MET(4)                                                    ILE(9)71    ARG(7)      ARG(16)           ARG(2)                 ALA(1)                      VAL(4)                           ARG(6)                                VAL(6)                                     ARG(3)                                          LS(4)     ARG(9)73    ASN(1)      THR(3)               ASP(3)78    LEU(4)      LEU(7)           TYR(9)                 AL(1)                      ALA(1)                           VAL(2)                                ALA(1)                                     LEU(6)                                          VAL(4)                                               PHE(5)                                                    LEU(5)80                              LEU(1)82              LEU(2)                         MET(1)                                               LEU(1)86                              ASP(2)92                    CYS(1)         CYS(1)    CYS(1)93    ALA(4)      ALA(5)     LEU(2)    THR(3)                                ALA(1)                                     THR(4)                                          ALA(4)                                               ALA(1)                                                    ALA(3)94    ARG(38)      ARG(24)           ASN(11)                 HIS(2)                      ARG(30)   ARG(23)                                     ARG(14)                                          ARG(30)                                               ARG(22)                                                    ARG(27)103   TRP(5)      TRP(9)          TRP(2)                           TRP(2)                                TRP(5)    TRP(2)                                               TRP(4)                                                    TRP(4)__________________________________________________________________________

The FRs, that contact FRs in the opposite domain and which presumably are the ones mainly responsible for the quaternary structure of the FV domains are listed in Table 4 below.

                                  TABLE 4__________________________________________________________________________Framework Residues That Contact FrameworkResidues in the Opposite Domain in Fabs ofKnown Three-Dimensional StructureANTIBODYPOSITION J539 McPC603           HyHEL-10                 HyHEL-5                      R19.9                           4-4-20                                36-71                                     B13I2                                          D1.3 NEWM KOL__________________________________________________________________________IN VL :36    TYR(3)      TYR(4)           TYR(3)                 TYR(5)    TYR(11)                                TYR(7)                                     TYR(1)                                          TYR(7)    TYR(5)38    GLN(10)      GLN(4)           GLN(9)                 GLN(5)                      GLN(5)                           GLN(3)                                GLN(6)                                     GLN(12)                                          GLN(6)                                               GLN(7)                                                    GLN(8)43    SER(7)      PRO(1)           SER(8)                 SER(5)                      THR(3)         SER(3)                                          SER(2)                                               ALA(5)                                                    ALA(1)44    PRO(10)      PRO(14)           PRO(8)                 PRO(11)   PRO(7)                                ILE(20)                                     PRO(16)                                          PRO(16)                                               PRO(7)                                                    PRO(13)46    PRO(3)85              MET(2)     THR(5)         VAL(1)    ASP(12)87    TYR(6)      TYR(4)           PHE(6)                 TYR(2)         PHE(5)                                     TYR(10)                                          TYR(8)                                               TYR(6)                                                    TYR(6)98    PHE(11)      PHE(8)           PHE(7)                 PHE(12)                      PHE(12)                           PHE(12)                                PHE(8)                                     PHE(13)                                          PHE(12)                                               PHE(10)                                                    PHE(15)100        ALA(2)IN VH :37    VAL(5)    ILE(2)                 VAL(1)                      VAL(4)                           VAL(2)                                VAL(1)                                     VAL(2)                                          VAL(4)                                               VAL(1)                                                    VAL(4)39    GLN(10)      GLN(4)           LYS(8)                 GLN(5)                      GLN(5)                           GLN(3)                                GLN(6)                                     GLN(10)                                          GLN(6)                                               GLN(4)                                                    GLN(7)43              ASN(4)     GLN(7)         LYS(6)    ARG(19)44         ARG(2)45    LEU(13)      LEU(12)           LEU(8)                 LEU(14)   LEU(8)                                LEU(11)                                     LEU(13)                                          LEU(14)                                               LEU(11)                                                    LEU(16)47    TRP(1)    TYR(2)     TRP(2)              TRP(3)                                               TRP(2)91    TYR(6)      TYR(4)           TYR(3)                 TYR(8)                      PHE(3)                           TYR(2)                                PHE(4)                                     TYR(3)                                          TYR(5)                                               TYR(3)103   TRP(11)      TRP(15)           TRP(16)                 TRP(11)                      TRP(4)                           TRP(18)                                TRP(24)                                     TRP(22)                                          TRP(19)                                               TRP(8)                                                    TRP(19)105   GLN(5)__________________________________________________________________________

The buried, inward-pointing FRs in the VL domains, i.e., those which are located in the domain interior, are listed in Table 5 below.

                                  TABLE 5__________________________________________________________________________Inward-Pointing, Buried Framework Residues inthe VL of Fabs of Known Three-Dimensional StructureANTIBODYPOSITION J539    McPC603         HyHEL-10               HyHEL-5                    R19.9                        4-4-20                            36-71                               B13I2                                   D1.3                                      NEWM                                          KOL__________________________________________________________________________ 2    ILE    ILE  ILE   ILE  ILE VAL ILE                               VAL ILE 4    LEU    MET  LEU   LEU  MET MET MET                               MET MET                                      LEU LEU 6    GLN    GLN  GLN   GLN  GLN GLN GLN                               GLN GLN                                      GLN GLN11    THR    LEU  LEU   MET  LEU LEU LEU                               LEU LEU                                      VAL ALA13    ALA    VAL  VAL   ALA  ALA VAL ALA                               VAL ALA19    VAL    VAL  VAL   VAL  VAL ALA VAL                               ALA VAL                                      VAL VAL21    ILE    MET  LEU   MET  ILE ILE ILE                               ILE ILE                                      ILE ILE23    CYS    CYS  CYS   CYS  CYS CYS CYS                               CYS CYS                                      CYS CYS35    TRP    TRP  TRP   TRP  TRP TRP TRP                               TRP TRP                                      TRP TRP37    GLN    GLN  GLN   GLN  GLN LEU GLN                               LEU GLN                                      GLN GLN47    TRP    LEU  LEU   TRP  LEU LEU LEU                               LEU LEU                                      LEU LEU48    ILE    ILE  ILE   ILE  VAL ILE ILE                               ILE VAL    ILE49                                         PHE58    VAL    VAL  ILE   VAL  VAL VAL VAL                               VAL VAL    VAL61    ARG    ARG  ARG   ARG  ARG ARG ARG                               ARG ARG    ARG62    PHE    PHE  PHE   PHE  PHE PHE PHE                               PHE PHE                                      PHE PHE71    TYR    PHE  PHE   TYR  TYR PHE TYR                               PHE TYR                                      ALA ALA73    LEU    LEU  LEU   LEU  LEU LEU LEU                               LEU LEU                                      LEU LEU75    ILE    ILE  ILE   ILE  ILE ILE ILE                               ILE ILE                                      ILE ILE78    MET    VAL  VAL   MET  LEU VAL LEU                               VAL LEU                                      LEU LEU82    ASP    ASP  ASP   ASP  ASP ASP ASP                               ASP ASP                                      ASP ASP83                                      PHE84    ALA    ALA        ALA  ALA     ALA       ALA THR86    TYR    TYR  TYR   TYR  TYR TYR TYR                               TYR TYR                                      TYR TYR88    CYS    CYS  CYS   CYS  CYS CYS CYS                               CYS CYS                                      CYS CYS102   THR    THR  THR   THR  THR THR THR                               THR THR                                      THR THR104   LEU    LEU  LEU   LEU  LEU LEU LEU                               LEU LEU                                      LEU VAL106   LEU    ILE  ILE   ILE          ILE                               ILE    VAL VAL__________________________________________________________________________

Those in the VH domain are listed in Table 6 below.

                                  TABLE 6__________________________________________________________________________Inward-Pointing, Buried Framework Residues inthe VH of Fabs of Known Three-Dimensional StructureANTIBODYPOSITION J539    McPC603         HyHEL-10               HyHEL-5                    R19.9                        4-4-20                            36-71                               B13I2                                   D1.3                                      NEWM                                          KOL__________________________________________________________________________ 2    VAL    VAL  VAL                VAL                               VAL VAL    VAL 4    LEU    LEU  LEU   LEU  LEU LEU LEU                               LEU LEU                                      LEU LEU 6    GLU    GLU  GLU   GLN  GLU GLU GLN                               GLU GLU                                      GLN GLN 9            PRO12    VAL    VAL  VAL   MET  VAL VAL VAL                               VAL VAL                                      VAL VAL18    LEU    LEU  LEU   VAL  VAL MET VAL                               LEU LEU                                      LEU LEU20    LEU    LEU  LEU   ILE  MET LEU MET                               LEU ILE                                      LEU LEU22    CYS    CYS  CYS   CYS  CYS CYS CYS                               CYS CYS                                      CYS CYS24    ALA    THR  VAL   ALA  ALA ALA ALA                               ALA VAL                                      VAL SER27    PHE    PHE  ASP   TYR  TYR PHE TYR                               PHE PHE                                      THR PHE29    PHE    PHE  ILE   PHE  PHE PHE PHE                               PHE LEU                                      PHE PHE36    TRP    TRP  TRP   TRP  TRP TRP TRP                               TRP TRP                                      TRP TRP38    ARG    ARG  ARG   LYS  LYS ARG LYS                               ARG ARG                                      ARG ARG40                           SER46       GLU  GLU   GLU48    ILE    ILE  MET   ILE  ILE VAL ILE                               VAL LEU                                      ILE VAL49       ALA                 ALA    ALA        ALA66    LYS    ARG  ARG   LYS             ARG ARG                                      ARG ARG67    PHE    PHE  ILE   ALA  THR PHE THR                               PHE LEU                                      VAL PHE69    ILE    VAL  ILE   PHE  LEU ILE LEU                               ILE ILE                                      MET ILE71    ARG    ARG  ARG   ALA  VAL ARG VAL                               ARG LYS                                      VAL ARG76                           SER78    LEU    LEU  TYR   ALA  ALA VAL ALA                               LEU VAL                                      PHE LEU80    LEU    LEU  LEU   MET  MET LEU MET                               LEU LEU                                      LEU LEU82    MET    MET  LEU   LEU  LEU MET LEU                               MET MET                                      LEU MET 82C  VAL    LEU  VAL   LEU  LEU LEU LEU                               LEU LEU                                      VAL LEU86    ASP    ASP  ASP   ASP  ASP ASP ASP                               ASP ASP                                      ASP ASP88    ALA    ALA  ALA        ALA     ALA                               ALA ALA                                      ALA90    TYR    TYR  TYR   TYR  TYR TYR TYR                               TYR TYR                                      TYR TYR92    CYS    CYS  CYS   CYS  CYS CYS CYS                               CYS CYS                                      CYS CYS94    ARG    ARG  ASN   HIS  ARG     ARG                               ARG ARG                                      ARG ARG107   THR    THR        THR  THR THR THR                               THR THR                                      SER THR109   VAL    VAL  VAL   LEU  LEU VAL LEU                               LEU LEU                                      VAL VAL111   VAL    VAL  VAL   VAL  VAL VAL VAL                               VAL VAL                                      VAL VAL__________________________________________________________________________

From the above, it may be seen that

(1) There are many FRs that either contact the CDRs or the opposite domain, or are found in the domain interior.

(2) These FRs, which could influence the structure of the combining site, and thus the antigen-binding characteristics of an antibody, are different from antibody to antibody.

It is obvious from these results that no one structure can serve as the perfect and sole basis of all "animalization", or in the present example "humanization", protocols. In fact, to "humanize" the 9 murine antibodies, shown in Table 1 above, by CDR-grafting, with a view to preserving their ligand-binding properties, the FRs listed in Table 2 to 6 above would have to be retained.

A search through the tables of immunoglobulin sequences (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed. US Dept. of Health and Human Service, NIH Publication No.91-3242 (1991)), shows that human variable domain sequences are known that already have most of the FRs that need to be preserved as shown in Table 7 below.

                                  TABLE 7__________________________________________________________________________Human Antibodies that are Most Similar inSequence to Murine Antibodies of KnownThree-Dimensional StructureANTIBODY DOMAIN   MOST SIMILAR HUMAN SEQUENCE__________________________________________________________________________HyHEL-10 VH       58P2'CL (77/112) VH FRAMEWORK          15P1'CL, ML1'CL (62/87) VH IMPT  58P2'CL, Ab26'CL, C6B2'CL (28/38) VL       IARC/BL41'CL (73/107) VL FRAMEWORK          IARC/BL41'CL (59/80) VL IMPT  IARC/BL41'CL (30/37)HyHEL-5 VH       ND'CL (74/116) VH FRAMEWORK          783c'CL, X17115'CL (63/87) VH IMPT  21/28'CL, 51P1'CL, 783c'CL, 8310'CL, AND, KAS, NEI'CL,          X17115'CL (25/37) VL       HF2-1/17'CL, KAS (65/105) VL FRAMEWORK          HF2-1/17'CL (57/80) VL IMPT  BI, DEN, HF2-1/17'CL, KUE, REI, WALKER'CL, WIL(=) (27/36)R19.9 VH       21/28'CL (73/119) VH FRAMEWORK          21/28'CL, 51P'CL, AND, LS2'CL, NEI'CL (60/87) VH IMPT  21/28'CL, 8E10'CL, LS2'CL (28/38) VL       WALKER'CL (78/107) VL FRAMEWORK          RZ (62/80) VL IMPT  RETI, WALKER'CL (33/36)4-4-20 VH       30P1'CL (77/116) VH FRAMEWORK          2P1'CL, 3D6'CL (65/87) VH IMPT  4B4'CL, M26'CL (36/41) VL       RPM1-6410'CL (91/112) VL FRAMEWORK          GM-607-'CL (68/80) VL IMPT  CUM, FR, NIM (33/36)J539  VH       30P1'CL, Vh38C1.10'CL (81/118) VH FRAMEWORK          18/2'CL, 30P1'CL, M43 (71/87) VH IMPT  38P1'CL, 56P1'CL, M72, M74 (36/40) VL       PA (62/105) VL FRAMEWORK          LEN, WEA (53/80) VL IMPT  BI, DEN, KUE, REI, WALKER'CL, WIL(=) (26/35)McPC603 VH       M72 (81/120) VH FRAMEWORK          4G12'CL, Ab18'CL, M72 (70/87) VH IMPT  56P1'CL, M72, M74, RF-SJ2'CL (36/42) VL       FK-001'CL, LEN (91/113) VL FRAMEWORK          LEN (70/80) VL IMPT  LEN (38/42)36-71 VH       21/28'CL (74/119) VH FRAMEWORK          21/28'CL, 51P1'CL, 783c'CL, AND 'CL, NEI'CL, X17115'CL,          (61/87) VH IMPT  21/28'CL, 8E10'CL (28/38) VL       AG (76/105) VL FRAMEWORK          RZ (63/80) VL IMPT  REI, RZ, WALKER'CL (34/37)B13I2 VH       56P1'CL (83/119) VH FRAMEWORK          4B4'CL, 4G12'CL, M26'CL, M72, RF-SJ2'CL, Vh38C1.10'CL          (68/87) VH IMPT  56P1'CL, M72, M74, RF-SJ2'CL (37/39) VL       RPMI-6410'CL (86/112) VL FRAMEWORK          GM-607-'CL (69.80) VL IMPT  CUM, NIM (36/39)D1.3  VH       C6B2'CL (72/116) VH FRAMEWORK          C6B2'CL (62/87) VH IMPT  M60'CL (32/37) VL       BR (75/107) VL FRAMEWORK          HF2-1/17'CL (64/80) VL IMPT  3D6'CL, BI, DEN, EU, KUE, PA, REI, WALKER'CL, WIL(=)          (32/36)__________________________________________________________________________

These human sequences are not necessarily those which are most similar to the murine antibodies, overall or in the framework regions only, the latter sequences being included in Table 7 above.

The number of murine amino acids that still need to be retained in order to have all the important FRs in the "humanized" or analogue versions of the murine antibodies, as shown in Table 7 above, ranges from 21 (for HyHEL-5:12 in VH and 9 in VL) to 5 (for B1312:2 in VH and 3 in VL). These are not very many amino acids, considering that the resulting "humanized" or analogue molecules will probably retain most or all their ligand-binding characteristics. It is possible that there exist other human sequences that are even more similar to these murine domains that are not included in the compilation of Kabat, et al. (1991), supra. When more sequences become available these may also be incorporated to improve the pool of basic data available.

b) Choosing the best human framework to use in the "humanization" of an antibody when its structure is not known.

In the absence of a three-dimensional structure, the identification of the FRs that are crucial to maintain the combining site structure is not easily done. Nevertheless, some proposals may be made from the data shown in Tables 2 to 6 above that have been collected in Tables 8 and 9 below for the VL and VH domains.

                                  TABLE 8__________________________________________________________________________Framework Residues in VL That Probably Need to Be Preserved in Orderto Reproduce the LigandProperties of the Original Antibody__________________________________________________________________________ J539  ##STR1##McPC603 DIVMTQ....L.V.....V.M.C kssqsllnsgnqknfla WYQQ.... PP.LLIYHyHEL-10 DIVL.Q....L.V.....V.L.C rasq------signnlh WYQQ.... SP.LLIKHyHEL-5 DIVL.Q....M.A.....V.M.C sass-------svnymy WYQQ.... SP.RWIYR19.9 .IQMTQ....L.A.....V.I.C rasq------disnyln WYQQ.... T.KLLVY4-4-20 DVVMTQ....L.V.....A.I.C rasq-slvhsqqntylr WYLQ.... .PKVLIY36-71 DIQM.Q....L.A.....V.I.C rasq------dinnfln WYQQ.... .I.LLIYB13I2 .VLM.QT...L.V.....A.ISC rasg-tillsdgdtyle WYLQ.... SP.LLIYD1.3  DI.M.Q....L.A.....V.I.C rasg------nihnyla WYQQ.... SP.LLVY J539  ##STR2##McPC603 gastres .V.DRF....S.TDF.L.I..V...D.A.YYC qndhsyplt F.A.T.L.I.HyHEL-10 yasqsis .I..RF......T.F.L.I..V...D..MYFC qqsnswpyt F...T.L.I.HyHEL-5 dtsklas .V..RF........Y.L.I..M...D.A.YYC qqwgr-npt F...T.L.I.R19.9 ytsrlhs .V..RF.......DY.L.I..L...D.ATY.C qqgsttprt F...T.L...4-4-20 kvsnrfs .V.DRF......T.F.L.I..V...D...Y.C sqsthvpwt F...T.L...36-71 ftsrsqs .V..RF......TDY.L.I..L...D.A.YFC qqgnalprt F...T.L..B13I2 kvsnrfs .V.DRF......T.F.L.I..V...D..VYYC fqgshvppt F...T.L.I.D1.3  ytttlad .V..RF....S.T.Y.L.I..L...DF..YYC qhfwstprt F...T.L...__________________________________________________________________________

                                  TABLE 9__________________________________________________________________________Framework Residues in VH That Probably Need to Be Preserved inOrder to Reproduce the Ligand Properties of the Original__________________________________________________________________________Antibody J539  ##STR3##McPC603 .V.L.E.....V.....L.L.C.T..FTF. dfyme WVRQ....RLEWIAHyHEL-10 .V.L.E..P..V.....L.L.C.V..D.IT sdyws WIRK...N.LEYM.HyHEL-5 ...L.Q.....M.....V.I.C.A..YTF. dywie WVKQR....LEWI.R19.9 .V.L.E.....V.....V.M.C.A..YTFT sygvn WVKQ...Q..EWI.4-4-20 ...L.E.....V.....M.L.C.A..FTFS dywmn WVRQS....LEWVA36-71 EV.L.Q.....V.....V.M.C.A..YTF. sngin WVKQ.....LEWI.B13I2 .V.L.E.....V.....L.L.C.A..FTP. rcams WVRQ...K.L.WVAD1.3  .V.L.E.....V.....L.I.C.V..F.L. gygvn WVRQ.....LEWL. J539  ##STR4##McPC603 asrnkgnkytteysasvkg RFIV.R.T....L.L.M..L...D.A.YYCARHyHEL-10 yvs---ysgstyynpslks RI.I.R......Y.L.L..V...D.A.YYC.NHyHEL-5 eilp--gsgstnyherfkg KA.F.A......A.M.L..L...D...YYCLHR19.9 yinp--gkgylsynekfkg .TTL.V......A.M.L..L...D.A.YFC.R4-4-20 qirnkpynyetyysdsvkg RFTI.R.D..S.V.L.M..L...D...YYCT.36-71 ynnp--gngyiaynekfkg .T.L.V......A.M.L..L...D.A.YFCARB13I2 giss--ggsytfypdtykg RF.I.R......L.L.M..L...D.A.YYCTRD1.3  miw---gdgntdynsalks RL.I.K......V.L.M..L...D.A.YYCAR J539  ##STR5##McPC603 nyygstwyf----dv W...T.V.V..HyHEL-10 wdg----------dy W.....V.V..HyHEL-5 gnydf--------dg W...T.L.V..R19.9 sfyggsdlavyyfds W...T.L.V..4-4-20 syygm--------dy W...T.V.V..36-71 seyyggsykf---dy W...T.L.V..B13I2 yssdpfyf-----dy W...T.L.V..D1.3  erdyrl-------dy W...T.L.V..__________________________________________________________________________

From Tables 8 and 9 above, it may be seen that many of the important FRs flank the CDRs. Among these flanking positions are most of the FRs that are involved in the contact with the opposite domain as shown in Table 4 above, and many of those which are in contact with the CDRs as shown in Tables 2 and 3 above. Moreover, almost all of the FRs that have been observed to participate in the binding to antigen (Amit, A. G., et al., Science 233:747-753 (1986); Sheriff, et al., P.N.A.S. (USA) 82:1104-1107 (1987); Padlan, E. A., et al., P.N.A.S. (USA) 86:5938-5942 (1989); Tulip, et al., Cold Spring Harbor Symp. Quant. Biol. 54:257-263 (1989); Bentley, et al., Nature (London) 348:254-257 (1990)), are in these flanking regions. Thus, during "animalization" or "humanization" or formation of the analogue peptides, not just the CDRs are transplanted, but also some of the residues immediately adjacent to the CDRs. This provides a better chance of retaining more of the ligand-binding properties of the original antibody. The likelyhood of retaining the antigen binding properties of the original antibody is even greater if the first few amino acids in the NH2 -termini of both chains are also transplanted, since some of them are found to be in contact with CDRs as shown in Tables 2 and 3 above. Further, Tables 8 and 9 above also show many other framework positions that are deemed structurally important in all the cases examined here. The xenogeneic residues at those positions should probably be retained as well.

Alternatively, it may possible to reduce immunogenicity, while preserving antigen-binding properties, by simply replacing those exposed residues in the framework regions which differ from those usually found in human antibodies (Padlan, E. A. (1991), supra). This would "humanize" the surface of the xenogeneic antibody while retaining the interior and contacting residues which influence its antigen-binding characteristics. The judicious replacement of exterior residues should have little, or no, effect on the interior of the domains, or on the interdomain contacts. For example, the solvent accessibility patterns of the Fv s of J539, a murine IgA (k) and of KOL, a human IgG1 (λ) have been found to be very similar (Padlan, E. A. (1991), supra).

At present, more than 35 different Fab structures have been elucidated by X-ray diffraction analysis, although atomic coordinates for only 11 are currently in the Protein Data Bank as shown in Table 1 above. Most of the available structures have been analyzed to only medium resolution, some having been refined to only a limited extent. Eventually, atomic coordinates for more and better-refined structures will become available, so that the "important" FRs will be more easily assessed. This will improve the theoretical predictive record of the present method for determining the best mode for the analogue peptides.

As already indicated above, the specificity of an antibody depends on the CDR structures and sometimes, on some neighboring residues as well. These structures, in turn, depend on contacts with framework amino acids and on the interaction of the VL and VH domains. Thus, to ensure the retention of binding affinity, not only the CDR residues must be preserved, but also those FRs that contact either the CDR's or the opposite domain, as well as all buried residues, which give shape to the variable domains.

This design of the humanized versions of murine antibodies is reached in stages as follows.

1- Choice of a xenogeneic model of known structure.

2- Choice of the target species FR.

3- Identification of xenogeneic/target species differences.

4- Identification of important xenogeneic amino acids.

(1) Choice of a xenogeneic model of known structure

The VH and VL domains of an antibody of desired specificity are classified according to Kabat et al.(1991), supra. Then, an antibody of the same species may be chosen, whose structure has been determined, and whose variable regions belong to the same classes and subclasses. Modeling the xenogeneic antibody in question to such structure ensures maximal chance for success. This, however, is not absolutely necessary since the relative positions of the important amino acids do not vary considerably even in variable regions of different classes. Thus, with less than a perfect match this method may still be applied to design the analogues of this invention. Once the xenogeneic model is chosen, it may be applied to identify the locations of important residues in the xenogeneic antibody to be animalized (humanized). Tables 2, 3, 4, 5, 6, 8 and 9 indicate the positions of the important amino acids in several antibodies whose structures have been determined to a high resolution level.

(2) Choice of the target species FR

The target species framework should, ideally, be a consensus framework. That is, one that has a maximum number of amino acids in common with all human frameworks of the same class. This is important, because, the goal of humanization is to avoid an immunological response against the engineered analogue peptide.

The target species framework that is chosen is that which shares the greatest number of important amino acids with the original xenogeneic antibody. Thus, in chosing the target species (human) FR, the similarity between the important amino acids is more important that the overall similarity.

In practice, the sequences of the xenogeneic variable chains are aligned with the consensus sequences from all variable region classes of the target species and the number of differences in the amino acids that must be retained from the xenogeneic species are scored. The human consensus sequence(s) that score(s) the lowest number of differences is (are) then chosen. These are the best analogue peptide candidates. Others with low numbers that are higher than the above may also be suitable, and are placed in a reserve pool, and so forth. If there are too many differences in the chosen framework (e.g., more than 16), then the same alignment procedure using all tabulated human sequences may be repeated in order to find a specific human framework whose similarity with the xenogeneic sequence is maximized at the positions of the important amino acids. Thus, most preferably, the target species FR should be a consensus sequence. Next preferable would be a framework of a common target species (human) antibody, and finally, the framework of any target species (human) antibody.

(3) Identification of xenogeneic target species differences

The xenogeneic sequences are then aligned with the target species sequences and the positions of all amino acids that differ in the murine and in the human frameworks are tabulated. Such a table contains the maximum number of amino acids that can be changed toward the full "animalization" ("humanization") of the xenogeneic antibody (see, Tables 31 and 32 below). If all those changes were to be made, a so called CDR-grafted antibody would be obtained. That is, only the original CDRs would be retained from the murine antibody. In some cases, possibly, such CDR-grafted antibody may maintain the original binding affinity. In most instances, however, the affinity of a CDR-grafted antibody would be considerably less than that of the original xenogeneic antibody. In order to maximize the chances for conserving the original affinity, the identities of all important amino acids must be preserved.

(4) Identification of important xenogeneic amino acids

If the outlined approach to animalization (humanizing) an antibody is followed strictly, the amino acids that are correspondingly important in the model xenogeneic antibody chosen in step 1 are retained. In a more preferred approach, however, the amino acids that have been shown to occupy important positions in other antibodies of the same species or of the target species may also be retained and are therefore taken out from the group of candidates to be mutated. This preferred approach may be particularly appropriate when there is a chance that the amino acids in question could make contacts with the CDRs or with the opposite chains. Once the important xenogeneic amino acids are identified, the DNA sequence may be mutagenized to change all other amino acids, which for the most part occupy exposed positions.

The present method is exemplified for a murine antibody humanized with the intent of diminishing or avoiding a HAMA response upon its administration to humans. Murine and human antibodies, whose three-dimentional structures have been deduced to a high degree of resolution, were utilized as guidance in the choice of the amino acids to be substituted in order to humanize the particular murine antibody utilized. The method, however, may be applied more generally to transform antibodies from one species into a less immunogenic form to be administered to a second species, provided that adequate three-dimentional models are available for antibodies from those species. Information on other murine antibodies from a Data Bank were used to modify the BrE-3 antibody with human amino acids. Similarly, antibodies of other species besides mouse may also be utilized, their CDRs and other amino acids preserved and those amino acids not considered "important" replaced with human amino acids. Similarly, the above approach may be applied to the preparation of "animalized" antibodies for any animal species. This may be attained by substituting amino acids of the antibody target species into an antibody of another species in accordance with this invention.

Various peptide structures, such as CDRs, and analogue antibodies, Fab, Fab', (Fab')2, and variable fragments having a desired specificity, may be constructed and optionally bridged via a linker. In addition, one or more of the peptides may be attached to one or more effector agent(s) or bridged via a linker. Multiple antibody, variable regions, Fab, Fab', (Fab')2, CDRs and the like, and combinations thereof, may also be constructed and bridged via linkers or attached to one or more effector agents such as are described below.

The cDNAs encoding the analogue variable regions of an antibody of a desired specificity may be cloned into a vector, optionally containing sequences encoding constant regions or fragments thereof, enzymes, neuropeptides, other peptide transmitters, toxins, hormones, operative conjugation regions, cytokines, lymphokines and the like, optionally under the same promoter. Although this is the cloning strategy utilized in the examplary disclosure of this invention, other methods known in the art may also be utilized such as co-expression and the like. In the exemplary disclosure provided herein, a BrE-3 mouse-human chimeric antibody specifically binding to human mammary mucin and carcinoma cells was constructed by joining the DNAs of the murine BrE-3 variable domain to a human constant domain (an effector agent) cloned into a hybrid vector, and the product expressed by transfecting the vector into myeloma cells. The variable regions of the chimeric antibody were modified at the DNA level to obtain an analogue or "humanized" chimeric polypeptide. The modifications to the variable regions of the peptides may be conducted by PCR amplification with primers that are custom tailored to produce the mutations desired.

The analogue "humanized" peptide prepared comprises the "humanized" variable regions of the BrE-3 mouse antibody (U.S. Pat. No. 5,075,219) and the kappa and gamma 1 constant region of a human antibody. This humanized antibody was characterized by its molecular weight and binding specificity, and shown to compete well with or better than the respective parent mouse and chimeric antibodies for the antigen with mouse antibody for the antigen. The analogue "humanized" peptide was shown to bind weakly to normal breast, lung, colon and endometrium, and strongly to carcinoma tissue sections by the ABC immunoperoxidase method. The portions of the CDR and FR regions of the non-modified peptide (mouse Fv regions) and effector agent (human Fc regions) were shown to be substantially identical to those of the non-human and human antibodies from which they were obtained. The analogue peptide and hybrid derivatives of this invention lacking any non-human constant region sequences possess less foreign antigenic epitopes than the whole xenogeneic antibodies from which they are derived. Accordingly, the inventors expect them to elicit a less complex immunogenic response in humans than the corresponding non-human whole antibodies or even than the chimeric antibodies. However, to what extent a portion of the non-human FRs may be deleted without altering the binding characteristics of the CDRs could not be predicted prior to this invention because of the substantial conformational alterations that normally occur upon modification of amino acid sequences in the interior regions that may affect the binding of the CDRs to the antigen.

The present invention, thus, provides a substantially pure, isolated analogue peptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or released by the cell, the polypeptide being selected from the group consisting essentially of at least one CDR or variable region of the light or heavy chains of an antibody of a first species having affinity and specificity for an antigen found on the surface or the cytoplasm of a carcinoma cell or released by the cell wherein about 1 to at least 46 amino acids in the FRs are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of a second species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids, combinations thereof, wherein each analogue peptide is operatively linked to at least one other analogue peptide, combinations thereof and mixtures thereof.

A single unit of the analogue peptide of the invention may be as short as the shortest CDR and as long as the longest combination of variable regions, antibodies, and the like, including non-peptide polymers of up to about 106 molecular weight, and in some instances even larger. When several units are linked or other combinations provided, the size of the analogue peptide increases accordingly. The smaller molecular weight analogue peptides are particularly suited for greater penetration of cells, the brain-blood barrier, and tumors, among others, whereas the higher molecular weight polypeptides are better suited for in vitro or in vivo imaging and diagnosis.

The analogue peptide of the invention may contain amino acid sequences derived from light and/or heavy chains of antibodies of a first or xenogenic species raised against a variety of antigens and/or epitopes. For example, the mouse antibodies disclosed in the examples were raised against human mammary fat globule mucin (BrE-3) and the "KC-4" antigen in human carcinoma cells (KC-4). Other antigens comprising a variety of epitopes may also be utilized as long as the antibody contributing the variable region displays affinity and specificity for human carcinomas that will permit their specific binding to carcinoma cells in a variety of tissues. Similarly, the antibodies may be raised in animals of different xenogeneic species. The antibody from which the polypeptide of the invention is derived may be a mouse, rat, goat, birds including poultry, rabbit, guinea pig, horse, bovine, and primate including human and simian antibody, among others. The preparation of the antibody and fragments thereof encompassed by the invention is similar, whether the origin of the antibody is human or non-human. The original mRNA is obtained from cells of the desired xenogeneic species but the remainder of the work-up is similar, utilizing a model antibody of the same xenogeneic species and substituting amino acids from the target species.

The humanization procedure described here is designed to minimize potential losses in antigen binding affinity that may result from the introduced amino acid changes. In the case of BrE-3, therefore, eight amino acid changes are made in the variable light region and in the variable heavy region. Furthermore, to minimize the immunological response to the humanized antibody, target human amino acid sequences were used that comprise the consensus sequences of all appropriate human variable regions. Nevertheless, neither the proposed amino acid changes nor the proposed human target sequences are necessarily the only choices available. Thus, many other individual amino acid changes and permutations thereof may be made without significantly affecting either the affinity of the resulting antibody or its human immunogenicity.

The following Tables 10 and 11 indicate other possible amino acid changes for BrE-3. The amino acid positions (or numbers) are as conventionally accepted (Kabat et al., 1991, supra). The most preferred changes are indicated under the heading "Most Preferred Analogue". If antibodies other than BrE-3 are to be considered, then the amino acids shown in the BrE-3 column would also be part of this group of most preferred choices. Amino acid changes that are not part of the most preferred group but that are still acceptable are indicated in the next column under the heading "Preferred Analogue". In some instances, the "Preferred Analogue" choices become too numerous and the least acceptable choices are provide instead for that position. Clearly, all amino acids other than those listed may be substituted at that position. These are shown under the last heading of "Not Preferred Analogue".

              TABLE 10______________________________________Alternative Amino Acids for VL Chain        MOST        PREFERRED  PREFERRED NOT PREFERREDVL   BrE-3   ANALOGUE   ANALOGUE  ANALOGUE______________________________________FR1   1D                          WIPLMCRT 2V     I          WKCRH 3V     L                    YC 4M     LVI        PTQ 5T 6Q 7T     IADS                 WYMCGH 8P     AE                   WYFKCN 9L     FP                   WYMH 10S    T                    HDQ 11L    NV                   WYCRGH 12P    S                    WIKH 13V                         YKCRHN 14S    T                    WMCE 15L    PFIL                 WYCGHDNEQ 16G 17D    TEQ                  WIYFPMCR 18Q    PS                   WYFCAHDN 19A    V                    WPKMCRTHNQ 20S                         WPLCHD 21I                         WPKCGDEQ 22S                         WVMCHE 23CFR2   35W 36F    YL         IVHN 37L    Q          WLVKRTHDE 38Q                         IFMCATSDN 39K    R                    WIPMCA 40S    P          FLKARTGQ 41G                         IYFPMCT 42Q                         WYVCD 43S    P                    WYFKMHDNEQ 44P                         WYKCRGHDQ 45K    EQR                  YPCGHD 46L    RV                   KCD 47L    V          WILMTSN 48I               FPLVMTS 49Y    S                    PLVMAFR3   57G               WVTSGDNEQ 58V               IYFLVMATQ 59P    S 60D    N                    WFMCR 61R    T 62F 63S    T          IYPLKARSG 64G    D 65S 66G                         YCHDQ 67S    A                    VKMCRHN 68E    GD         VMCARSGQ 69T                         SPMCE 70D                         WIFPMCR 71F                         WKMTEQ 72T                         WLMCGHNEQ 73L 74K    NLRE                 WLMCHD 75I    L 76S    IT                   LVMC 77R    S                    WYFLKCHQ 78V    ALI                  WYFKCRHNE 79E    KGQ                  WVKM 80A    P                    WLKM 81E               ILVKMAGDNE 82D 83L    MV                   WCRH 84G               LVARTSG 85V    IM                   WFKCQ 86Y 87F    YL         IMSHE 88CFR4   98F 99G100G    ASQ        IPVKRTG101G102T103K    NR         IYMATGHDEQ104L    V          LG105E               ILVTSGHNEQ106I               YLVKMRTD106aA              PLVTI     POSSIBLE                             A.A. INSERT107K    R          ILVMATSGNE______________________________________

                                  TABLE 11__________________________________________________________________________Alternative Amino Acids for VH Chain  MOST PREFERRED             PREFERRED                      NOT PREFERREDVL BrE-3  ANALOGUE   ANALOGUE ANALOGUE__________________________________________________________________________FR1    1E Q          PVLKARGHDEQ    2V M                   WYPKCRHV    3K QR         L    4L                     WYKCRSN    5E VD                  WYCG    6E QD                  WLYFMSH    7S T                   IMCRHD    8G E    9G                     WIYKCRNQ    10G  DA                  WIYFMCH    11L  VF                  WKCGHN    12V  I                   WYPTHD    13Q  KE                  WYFCD    14P                    WIYMCRDQ    15G                    IYCHN    16G  RSE                 WIYFMC    17S  PA                  WIYCHDNE    18M                    WYCD    19K  R                   WIYPCH    20L  V                   WYFPKDNQ    21S                    WMH    22C    23A  TSE                 WFLMCH    24A  V                   WKMCRHNEQ    25S                    WIVMRHDEQ    26G    27F                    SPMCR    28T  AINS                CHQ    29F                    WYKCRHDNEQ    30S                    WLMCDQFR2    36W    37V           WIFLVMATGQ    38R                    YFS    39Q                    FPVMCA    40S  VA                  WY    41P  TS         PLVARTSHNEQ    42E  G                   YPLKMCTHN    43K                    WIYLVCS    44G  SR                  WIFMCH    45L    46E  Q                   FPMCRTD    47W                    PMARNQ    48V  STG                 YPKCARTHNQ    49A                    WFPKCRHNQFR3    66R                    SYLVMCATSDE    67F                    WYPKMHNEQ    68T  IS                  WYPGE    69I                    WYKCHNQ    70S  L                   WVMCHDEQ    71R                    WYFCHDQ    72D  N          VKRTSGHDE    73D  N                   WYFLC    74S  A          FPLVTGDN    75K  EN                  NPCGD    76S  NTRK                WFPLMCE    77R  TNIVSM              WFC    78V  LA                  WKCRNE    79Y  FH                  WPG    80L                    WYLKCARGHEQ    81Q  E                   WYFPC    82a  SDN                 WMQ    82b  IR                  WYFPLMHQ    82c           PLVMAGE    83R  EKT                 WILCH    84A  SPVTI               WKCDEQ    85E  D          WIYFPLMCRG    86D    87T  M                   WLVMCNE    88G  A    89L  ITVM                WYPKGEQ    90Y  HF    91Y                    WIPLVKMAGQ    92C    93T  ASHAV               WIFR    94G  R          TSDQLAW   103W  A   104G  YH   105Q  THR                 IYFLVMCDQ   106G   107T  AQ   108L  STGM                WYCHDE   109V  LT         IK   110T  SL   111V   112S  T   113A  S          PLVATG__________________________________________________________________________

In one particularly preferred embodiment of the invention, the anti-human carcinoma analogue peptide comprises a member selected from the group consisting of amino acid sequence ID No. 67 to 73 of Table 47, sequence ID No. 75 to 81 of Table 48, and sequences wherein about 1 to at least 46 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in human antibodies, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to 10 amino acids, alone or with an N-terminal fragment of about 1 to 10 amino acids; CDR fragments thereof, or combinations thereof wherein each analogue peptide is operatively linked to at least one other analogue peptide, and mixtures thereof.

The present analogue peptides are provided either as a naked peptide or in glycosylated form. When provided in glycosylated form, the analogue peptide may be operatively linked to a glycosyl residue, provided by the eukaryotic cell where it is expressed, or it may be cloned and expressed in a prokaryotic cell as the naked polypeptide, and the glycosyl residues added thereafter, for example by means of glycosyl transferases known in the art. Examples of glycosyl residues that may be added to the analogue peptide of the invention are N-glycosylated and O-glycosylated residues, among others. The glycosyl residues added to the naked analogue peptide may have a molecular weight of about 20 to 50,000 daltons, and more preferably about 100 to 20,000 daltons or greater, depending on the size and molecular weight of the peptide to which they are attached. However, other types of polysaccharides and molecular weights may also be present. The glycosyl residues may also be attached to the naked analogue peptide of the invention by chemical means as is known in the art.

A single CDR is the smallest part of an antibody known to be capable of binding to an antigen. The sequences of the VL and VH CDRs of the exemplary analogue are shown in Tables 47 and 48 below. Thus, small peptides that have the sequence of a single CDR can bind antigen and are, therefore, suitable for imaging tumors in vivo. A CDR attached to an effector peptide may be chemically synthesized or encoded in a DNA segment. Such small molecules have great tumor penetration and extremely rapid clearing properties when compared to larger antibody fragments. In some cases it is more convenient to produce these small molecules by chemical synthesis as is known in the art rather than by fermentation. These small peptides are, in some cases, completely non-immunogenic, thus avoiding the HAMA response altogether. Also preferred are 2 and 3 CDR units per chain operatively linked to one another by 1 to at least 10 amino acids and up to the entire inter CDR sequence length as positioned in the variable regions.

Heavy and light chain analogue variable regions may be obtained individually or in VH /VL pairs, or attached to an effector peptide such as a constant region(s) or portions thereof, a drug, an enzyme, a toxin, a whole antibody, or any other molecule or radioisotope. The fragments of the analogue variable regions may be synthesized chemically as is known in the art or from the DNA segments encoding the non-human variable regions. This may be attained by PCR amplification of the DNA with primers synthesized to contain the desired mutation as is known in the art. Similarly, the fragments encoding analogue variable regions may be synthesized chemically or obtained by extablished cloning methods of restriction digestion, ligation, mutagenesis, and the like.

There are advantages to using different molecular variants of the analogue peptide depending on the specific applications for which they are intended, some of which are listed below.

Smaller molecules penetrate target tissues more efficiently and are cleared from the body much more rapidly than larger molecules.

Single chain molecules can be manipulated and synthesized more efficiently that multiple chain molecules.

Many of these variants can be synthesized efficiently and inexpensively in bacteria.

Bi-functional or multifuncitonal molecules may carry polypeptide effectors, such as enzymes, toxins, radioisotopes, drugs, and other molecules, to a target tissue.

The following list encompasses exemplary analogue peptides of the invention engineered with molecules derived from antibodies or antibody fragments. These analogue peptides, among others, are suitable for the practice of this invention. A more extensive list of polypeptide constructs may be found in O'Kennedy, R., and Roben, P. (O'Kennedy, R., and Roben, P., "Antibody Engineering: an Overview", Essays Biochem. (England) 26:59-75 (1991)).

The analogue peptides and hybrid peptides of this invention encompass CDRs and/or analogue variable regions, monoclonal antibodies, antibody fragments such as Fab, Fab', (Fab')2, and fragments thereof, CDRs, constant regions, single or multiple-domain and catalytic fragments, bi-functional or multifuncitonal combinations thereof, enzymes, peptide hormones, molecules such as drugs and linkers, transmitters, and toxins, among others. These are suitable for imaging, therapy, diagnostics, and biosensors.

Single-Chain Antigen-Binding Polypeptides

Single chain antigen-binding polypeptides and their syntheses have been described, e.g., by Bird, R. E., et al. (Bird, R. E., et al., Science 242(4877):243-6 (1988); Bird, R. E., et al., Science 244(4903):409 (1989)). For example, analogue peptides such as VH -linker-VL and VL -linker-VH, have significant advantages over monoclonal antibodies in a number of applications. These may be expressed and purified from E. coli. The polypeptide linker binding the two chains may be of variable lengths. For example, about 2 to 50 amino acid residues, and more preferably about 12 to 25 residues, and may be expressed in E. coli.

Single Chain Fv,(scFv or sFv)

These are single chain analogue peptides containing both VL and VH with a linker such as a peptide connecting the two chains (VL -linker-VH). The engineering may be done at the DNA level. So, knowledge of the sequence is required. These analogue peptides have the conformational stability, folding, and ligand-binding affinity of single-chain variable region immunoglobulin fragments and may be expressed in Escherichia coli. (Pantoliano, M. V., et al., Biochem. (US) 30 (42):10117-25 (1991)). The peptide linker binding the two chains may be of variable length, for example, about 2 to 50 amino acid residues, and more preferably about 12 to 25 residues, and may be expressed in E. coli. (Pantoliano, M. V., et al. (1991), supra). An analogue peptide such as an scFv may be expressed and prepared from E. coli and used for tumor targeting. The clearance profiles for scFv in some situations fragments are advantageous relative to those of normal antibodies, Fab, Fab' or (Fab')2 fragments. (Colcher, D., et al., J. Natl. Cancer Inst. 82 (14):1191-7 (1990)). Another type of analogue peptide comprises a VH -linker-VL and may have about 230 to 260 amino acids. A synthetic gene using E. coli codons may be used for expression in E. coli. A leader peptide of about 20 amino acids, such that of Trp LE may be used to direct protein secretion into the periplasm or medium. If this leader peptide is not naturally cleaved, the sFv analogue peptide may be obtained by acid cleavage of the unique asp-pro peptide bond placed between the leader peptide and the sFv -encoding region. (Huston, J. S., et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in E. coli.", P.N.A.S. (USA) 85 (16):5879-83 (1988)). The construction, binding properties, metabolism and tumor targeting of the single-chain Fv analogue peptides derived from monoclonal antibodies may be conducted as previously described. (Milenic, D. E., et al., Cancer Res. (US) 51 (23 pt1):6363-71 (1991); Yokota, et al., "Rapid Tumor Penetration of a single-chain Fv and Comparison with Other Immunoglobulin Forms", Cancer Res. (US) 52(12):3402-8 (1992)). This type of analogue peptide provides extremely rapid tumor penetration and even distribution throughout tumor mass compared to IgG or Ig fragments Fab and F(ab')2.

Bifunctional scFv -Fxn or Fxn-scFv

An example of this type of analogue peptide is a VL -linker-VH with an effector peptide such as a hormone, enzyme, transmitter, and the like. These hybrid analogue peptides may be prepared as described by McCarney, et. al. (McCarney, J. E. et al., "Biosynthetic Antibody Binding Sites: Development of a Single-Chain Fv Model Based on Antidinitrophenol IgA Myeloma MOPC 315", J. Protein Chem. (US) 10 (6):669-83 (1991)). A bi-functional hybrid analogue peptide containing an Fc -binding fragment B of staph protein A amino terminal to a single-chain analogue Fv region of the present specificity is also encompassed and may be prepared as previously described. (Tai, M. S., et al., Biochem. 29 (35):8024-30 (1990)). In this example of a hybrid analogue peptide of this invention is a Staph. A fragment B (anti Fc))-scFv polypeptide. The order is backward of normal cases. This FB-sFv may be encoded in a single synthetic gene and expressed as peptide in E. coli. This analogue peptide is a good example of a useful multifunctional targetable single-chain polypeptide. A hybrid analogue peptide also comprising antibodies to a human carcinoma receptor and angiogenin is also part of this invention. Angiogenin is a human homologue of pancreatic RNAse. This is an (Fab')2 -like antibody-enzyme peptide effector. Another hybrid analogue peptide comprising a VH -CH1 heavy chain-RNAse may be expressed in a cell that secretes a chimeric light chain of the same antibody. A secreted antibody of similar structure was shown to cause the inhibition of growth and of protein synthesis of K562 cells that express the human transferrin receptor.(Rybak, S. M., et al., "Humanization of Immunotoxins", P.N.A.S. 89:3165-3169 (1992)).

Bi-specific Antibodies

A monoclonal anti-Fc antibody may be incorporated into a bi-specific F(ab')2 analogue peptide as described by Greenman, J., et al. (Greenman, J., et al., Mol. Immunol. (Enlgand) 28 (11):1243-54 (1991). A bi-specific F(ab')2 comprising two (Fab'-(thioether-link)-Fab') chains has the advantage that it is not blocked by human Fc gamma RII antibody. Thus, these are also utilized as effector agents herein. Bi-specific antibodies may be obtained when two whole antibodies are attached. Another way to obtain bi-specific antibodies is by mixing chains from different antibodies or fragments thereof. In this manner the "left" branch of the bi-specific antibody has one function while the "right" branch has another. The analogue peptides in accordance with this invention may be screened with a filamentous phage system. This system may also be used for expressing any genes of antibodies or fragments thereof as well as for screening for mutagenized antibody variants as described by Marks, J. D., et al. (Marks, J. D., et al., "Molecular Evolution of Proteins on Filamentous Phage. Mimicking the Strategy of the Immune System", J.Mol. Biol. (England) 267 (23):160007-10 (1992)). A library of VH and Vk genes or analogue therof may be cloned and displayed on the surface of a phage. Antibody fragments binding specifically to several antigens may be isolated as reported by Marks. J. D., (Marks, J. D., "By-Passing Immunization. Human Antibodies from V-gene Libraries Displayed on Phage", J. Mol. Biol. (England) 222 (3):581-97 (1991)).

Covalent Oligosaccharide Modifications

The present analogue peptides alone or as hybrid peptides comprising antibodies and fragments thereof may be, e.g., covalently modified utilizing oxidized oligosaccharide moieties. The hybrid analogue peptides may be modified at the oligosaccharide residue with either a peptide labeled with a radioisotope such as 125 I or with a chelate such as a diethylenetriaminepentaacetic acid chelate with 111 In. The use of oligosaccharides provides a more efficient localization to a target than that obtained with antibodies radiolabeled either at the amino acid chain lysines or tyrosines (Rodwell, J. D. et al., "Site-Specific Covalent Modification of Monoclonal Antibodies: In Vitro and In Vivo Evaluations", P.N.A.S. (USA) 83:2632-6 (1986)).

Particularly preferred analogue peptides of this invention are those having the sequences ID Nos. 67 through 73, or 75 through 81, and analogues thereof, wherein about 1 to 42 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of the species for which use it is intended, such as human, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids, or combinations thereof. Examples of possible substitute amino acids are shown in Table 10 and 11 above, and are indicated in the columns titled BrE-3, Most Preferred Analogue and Preferred Analogue amino acids subtituents. Others are also suitable as may be deduced by the method described herein. These amino acid sequences may be bound by a peptide or non-peptide linker such as is known in the art. Examples of peptide linkers are polylysines, leucine zippers, EGKSSGSGSEJKVD, and (GGGGS)x3, and non-peptide polymers, among others. Effector agents such as peptides and non-peptides may also be attached to the analogue peptides of the invention. These include non-peptide polymers, monomers, atoms, etc. These are discussed below.

Another preferred embodiment comprises a bi-functional analogue peptide having a pair of light and heavy chains of the same specificity attached to one another by a linker, such as those provided above. In another preferred embodiment, a bi-functional analogue peptide comprises one set of light and heavy chains comprising at least one xenogeneic CDR or variable region, e.g., amino acid sequences ID Nos. 12 or 13 of Table 16 below, with the modifications shown above, wherein about 1 to 42 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of the target species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids, and one set of light and heavy chain comprising at least one xenogeneic CDR or analogue variable region, e.g., amino acid sequences ID Nos. 12 or 13 having a different set of substitute amino acids, wherein about 1 to at least 46 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of the target species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids, or fragments thereof, or combination thereof. Multi-functional hybrid analogue peptides may comprises several identical units or combinations of the above bi-functional analogue peptides of the same or different specificities or xenogeneic species. Preferred analogue peptides are those comprising murine CDRs and other regions substituted with human amino acids.

In another aspect, this invention provides a hybrid analogue peptide that comprises at least one anti-carcinoma analogue peptide and at least one effector agent operatively linked to the peptide, combinations thereof and mixtures thereof. The effector agent utilized in this invention comprises peptide polymers other than the constant region of an antibody of the same species as the CDRs, non-peptide polymers, monomers, and atoms such as metals. In one particularly preferred embodiment, the effector agent may comprise an atom such a radioisotope, an enzyme or a fluorescent label. These effector peptides are suited for in vivo and in vitro assays because they permit the identification of complexes formed by the peptide of the invention. Radioisotopes are particularly preferred for in vivo imaging. Polypeptide labeling is known in the art (Greenwood, F. C., et al., Biochem. J. 89:114-123 (1963)). When a glycosylated polypeptide is utilized, the radiolabel may be attached to the glycosyl residue as is known in the art (Hay, G. W. et al, in Methods in Carbohydrate Chemistry, Vol 5:357, Whistler, R. L. Ed., Academic Press, NY and London (1965)). Effector agents comprising a monomer may be therapeutic, immunogenic or diagnostic agents, radioisotopes, DNA, or RNA monomers, chemical linkers, transmitter molecules, combinations thereof, or combinations thereof with peptide and non-peptide polymers or copolymers and atoms. Examples of therapeutic agents are anti-neoplastic drugs such as vincristine, intercalation drugs, adriamycin, enzymes, toxins and hormones, among others. Examples of immunogenic agents are other vaccines for carcinomas or for others purposes. Examples of diagnostic agents are radioisotopes and enzymes, among others. Examples of therapeutic, immunogenic and diagnostic agents are toxins, vaccines, and radioisotopes, among others. Examples of radioisotopes are 111 In, 35 S, 90 Y, 186 Re 225 Ac 125I and 99 Tc, among others. Examples of DNA and RNA monomers are A, T, U, G, C, among others. Examples of chemical linkers are dithiobis(succinimidyl)propionate and bis-(sulfosuccinimidyl)suberate, among others. Examples of transmitter molecules are cAMP and cGMP, among others. Examples of toxins are ricin A-chain and abrin A-chain, among others.

When the effector agent is a non-peptide polymer linked to the analogue peptide of the invention it may comprise an ester, ether, vinyl, amido, imido, alkylene, arylalkylene, cyanate, urethane, or isoprene polymers, DNA polymers, RNA polymers, copolymers thereof and copolymers thereof with peptide polymers or monomers, or have labeled atoms attached thereto. Examples of these are polyesters, polyethers, polyethyleneglycols, polyvinyls, polyamido and polyimido resins, polyethylenes, polytetrafluoroethylene, poly(ethylene)terephathalate, polypropylene, silicone rubber, isoprenes and copolymers thereof, copolymers of silicone and carbonated polylactic or polyglycolic acid or collagen, and the like. Particularly preferred are biodegradable and bioresorbable or bioabsorbable materials, which if detached from the polypeptide and left in the systemic circulation will not damage endogenous tissues. The effector agent being a peptide may comprise antibodies such as IgG, IgM, IgE or IgD, the constant region of antibodies of a species different from the variable region or fragments thereof, and the CDRs, variable regions, Fab, Fab', (Fab')2 fragments of antibodies of the classes described above, hormones, enzymes, peptide trasnmitters and whole antibodies, combinations thereof, and combinations thereof with non-peptide polymers, copolymers, monomers and atoms such as radioisotopes. Examples of other antibodies, Fab, Fab', (Fab')2, CDRs and variable regions thereof are those that specifically bind carcinoma epitopes such as do BrE-3 and KC-4 and others having specificities for different carcinoma epitopes such as BrE-1, Br-E2, and Mc5, among others, and fragments thereof. Examples of peptide transmitters and hormones suitable for use herein are insulin, growth hormone, FSH, LH, endorphins, and TNF, among others. Examples of enzymes are peroxidase, LDH, alkaline phosphatase and galactosidase, among others.

In a particularly preferred embodiment, the analogue peptide of the invention comprises non-human CDR and variable region sequences, and the effector peptide comprises the constant region of the light or heavy chains of a human antibody or fragments thereof capable of binding γ-globulin, protein G or protein A, or fragments lacking this binding capability. Also preferred is a half humanized/half chimeric or murine antibody (e.g., humanized light chain and murine or chimeric heavy chain and viceversa). In one of the most preferred embodiments, the analogue peptide(s) comprise(s) all CDRs, flanking sequences of 1 to 10 amino acids connecting them, and an N-terminal region of at least up to 10 amino acids.

In another preferred embodiment, the hybrid analogue peptide comprises two heavy and two light chains, each light and heavy chain comprising at least one CDR or analogue variable region polypeptide or fragments thereof of one species and the constant region and the substitute amino acids of an antibody of a different species such as human, at least one other CDR, analogue variable region, chimeric Fab, Fab' or (Fab')2, fragments thereof, combinations thereof, and mixtures thereof. Still more preferred is a hybrid analogue peptide comprising at least two "humanized" murine-human or chimeric antibody fragments thereof, Fab, Fab' or (Fab')2 fragments thereof operatively linked to one another. The peptide fragments may be covalently attached to one another as is known in the art (Marchis-Mouren G., et al., "HT 29, a Model Cell Line: Stimulation by the Vasoactive Intestinal Peptide (VIP); VIP Receptor Structure and Metabolism", Bioch. 70 (5):663-71 (1988)), or they may be synthesized by methods known in the art (Allen, G., et al., "Production of Epidermal Growth Factor in Escherchia Coli from a Synthetic Gene", J. Cell Sci. Suppl. 3:29-38 (1985)).

In another preferred embodiment, the hybrid analogue peptide of the invention described above having two heavy and two light analogue chains operatively linked to one another, where each pair of heavy and light chains has specificity for a different epitope. One example of this analogue peptide is a pair of "humanized" variable region heavy and light chains of a BrE-3 analogue peptide and a pair of "humanized" variable region light and heavy chains of a KC-4 analogue peptide that are covalently attached to one another by a peptide or non-peptide polymer or a disulfide bridge, or non-covalently by means of a lencine zipper or two helical structures, and the like. Non-peptide polymers may be covalently attached to peptides by methods known in the art (Duronio, V., et al., "Two Polypeptides Identified by Interleukin 3 Cross-Linking Represent Distinct Components of the Interleukin 3 Receptor", Exp. Hematol. 20 (4):505-11 (1992)). In another embodiment, the invention provides a hybrid analogue peptide comprising at least one CDR or analogue variable region of the heavy chain of an antibody of a first species or fragments thereof, operatively linked to a first effector agent, and at least one CDR or analogue variable region of the light chain of an antibody of a second species or fragments thereof operatively linked to a second effector agent and combinations thereof, wherein each pair of light and heavy chains has a predetermined specificity, combinations thereof, and mixtures thereof. In another preferred embodiment of the hybrid analogue peptide, at least one CDR or analogue variable region of the heavy chain of a murine antibody or fragments thereof and at least one CDR or variable region of the light chain of a murine antibody or fragments thereof are linked to one another by a non-peptide polymer such as an isoprene polymer or monomer. In still another preferred embodiment, the hybrid analogue peptide of the invention is one wherein at least one pair of light and heavy chains comprising at least one murine CDR or analogue variable region or fragment therof is linked to at least one other pair of light and heavy chains comprising at least one murine CDR or analogue variable region or fragment thereof. In another embodiment the two or more Fv regions are covalently attached to one another by a peptide or non-peptide polymer or a disulfide bridge, or non-covalently by means of a leucine zipper or two helical structures, and the like. In a most preferred embodiment, the analogue peptides and hybrid polypeptides of the invention have affinity and specificity for an epitope located in the most hydrophilic region of a 20 amino acid tandem repeat that makes up a large part of the polypeptide core of mammary mucin, to hexamer fragments of the sequence APDTRPAPG or trimer TRP fragments shown to afford the strong binding of all five different monoclonal antibodies raised against human mammary fat globule (Mc1, Mc 5, BrE-1, BrE-2 and BrE-3). The monoclonal antibodies were shown to bind to different but overlapping polypeptide epitopes but to have different tissue and tumor specificities, to quantitatively differ in their binding to breast carcinoma cell lines when observed by flow cytometry and have different competition patterns for binding to the native antigen on breast carcinoma cells. Also preferred amongst antibodies utilized for the preparation of the present analogue peptide and hybrid polypeptide are those that exhibit strong binding to the hexamer peptides described above or to fragments comprising a TRP trimer to tandem repeats thereof. In one most preferred embodiment, the analogue peptide comprises the humanized antibodies expressed by the hybridoma cell lines having the ATCC Accession No. HB 11200 (BrE-3 HZ). This cell was deposited with the ATCC under the Budapest Treaty on Nov. 13, 1992.

This invention also provides an anti-human carcinoma composition that comprises the anti-carcinoma analogue peptide and/or hybrid polypeptide of the invention and a pharmaceutically-acceptable carrier. The anti-human carcinoma analogue peptide and hybrid peptide provided herein may be present in the composition in an amount of about 0.001 to 99.99 wt %, more preferably about 0.01 to 20 wt %, and still more preferably about 1 to 5 wt %. However, other amounts are also suitable. Pharmaceutically-acceptable carriers are known in the art and need not be further described herein and may be provided in a separate sterile container or in admixture with the polypeptide. Typically, saline, aqueous alcoholic solutions, albumin-saline solutions, and propylene glycol solutions are suitable. However, others may also be utilized. When utilized for therapeutic purposes, the composition may also contain other ingredients as is known in the art. Examples of these are other anti-neoplastic drugs such as adriamycin, and mitomycin, among others, cytoxan, PALA and/or metrotrexate. However, other therapeutic drugs, diluents, immunological adjuvants and the like may be also be added. When the composition described above is utilized for in vivo imaging, it may comprise about 0.001 to 99.9 wt % analogue peptide, and more preferably about 0.01 to 25 wt % analogue peptide. Typically, when the composition is utilized for therapeutic purposes it may contain about 0.001 to 99.9 wt % analogue peptide, and more preferably about 0.01 to 30 wt % analogue peptide. When utilized for the in vitro diagnosis of carcinomas the composition of the invention may comprise about 0.001 to 35 wt % analogue peptide, and more preferably about 0.001 to 10 wt % analogue peptide. Other amounts, however, are also suitable.

Such products find one utility in the treatment of carcinomas, such as breast, lung, ovary, endometrial, pancreas, prostate and colon cancers, among others. The "humanized", "half humanized" and "partially humanized" analogue peptides may be used for treatment or diagnosis of humans. The "animalized", "half animalized" and "partially animalized" analogue peptides of the invention may be utilized for the treatment of species such as were described above so far as the amino acids of such species are substituted for those of the xenogeneic amino acids and any constant region present in the analogue. The present analogue peptides are particularly suitable for repeated administration to a subject and for long term therapies such as is the case of metastases and/or reoccurrence of tumors.

Also provided herein is a diagnostic kit for carcinoma, that comprises the composition of the invention comprising the anti-carcinoma analogue peptide, a solid support, anti-carcinoma antibody, γ-globulin, protein G or protein A, and instructions for its use. This diagnostic kit may be utilized by covalently attaching the antigen or analogue peptide of the invention to the solid support by means of a linker as is known in the art. In a particularly preferred embodiment, the support is coated with methylated albumin as described in U.S. Pat. No. 4,572,901, the relevant text of which being incorporated herein by reference. When a biological sample is added to a well, the analogue peptide of the invention will bind to carcinoma antigen present in the biological sample. If a competitive assay is utilized, to the solid supported antigen are added a known amount of the analogue peptide and the sample. Thereafter, labeled y-globulin, protein G or protein A in labeled form may be added for detection. Anti-carcinoma antibodies of a first species may be obtained by challenging a subject of another species with carcinoma cells, the human milk fat globule mucin and the like, as is known in the art (Peterson, J. A., et al., Hybridoma 30 9:221 (1990)). Monoclonal antibodies may be prepared as described by Kohler and Milstein (Kohler, G. and Milstein, C., "Continuous Culture of Fused Cell Secreting Antibody of Predefined Specificity", Nature 256:495-497 (1975)). Suitable for use in this invention are antibodies such as IgG, IgM, IgE and IgD. Protein A, protein G and γ-globulin may be obtained commercially.

Still part of this invention is a diagnostic kit for human carcinomas that comprises the anti-carcinoma composition comprising a hybrid analogue peptide and an effector agent comprising an enzyme, a radioisotope, a fluorescent label and/or a peptide comprising the constant region of an antibody of the species for which use it is intended, or fragments thereof capable of binding γ-globulin, protein G or A, anti-human carcinoma antibody, γ-globulin, protein G or protein A, a solid support having operatively linked thereto an antigen which specifically binds to the anti-carcinoma hybrid analogue peptide of the invention and the antibody, and instructions for its use. When the effector agent comprises a peptide, such as the constant region of an antibody of the target species, the solid support may have operatively linked thereto an antibody which specifically binds to a portion of a fusion protein other than the antigen of the invention. This permits the binding of the anti-carcinoma analogue peptide to the antigen molecule now attached to the solid support. Any complex formed between the hybrid analogue peptide of the invention and the supported carcinoma antigen will, thus, remain attached to the solid substrate. A competitive assay may then be conducted by addition to the solid supported antigen of a known amount of the hybrid antigen peptide and the sample. The amount of antigen present in the sample may be obtained from a dilution curve by addition of γ-globulin, protein G or protein A, e.g., labeled, to bind the hybrid analogue peptide that is now attached to the support. This kit may be used in a competitive assay where the supported antigen molecule competes with antigen in the sample for a known amount of the analogue peptide of the invention. The assay was described by Ceriani, R. L., et al. (Ceriani, R. L., et al., Anal. Biochem. 201:178-184 (1992)), the relevant text thereof being incorporated herein by reference.

Still part of this invention is an in vivo method of imaging and/or diagnosing a carcinoma, that comprises administering to a subject suspected of carrying a carcinoma the anti-carcinoma analogue peptide of the invention in radiolabeled form, in an amount effective to reach the carcinoma and bind thereto, and detecting any localized binding of the labeled analogue peptide to the tumor. Typically, the analogue peptide of the invention may be administered in an amount of about 0.01 to 5000 μg/kg weight per treatment, and more preferably about 0.1 to 500 μg/kg weight per treatment. However, other amounts may also be utilized. Radiolabels that may be utilized are 111 In, 35 I, 99m Tc, and 131 I, among others. These radioisotopes may be detected with a PET scanner, NMR imaging, and radioactivity counting apparatus that are in wide use by the medical community.

Also provided herein is an in vitro method of diagnosing a carcinoma that comprises contacting a biological sample with the anti-carcinoma analogue peptide or hybrid polypeptide of the invention to form an anti-carcinoma analogue peptide-antigen complex with any carcinoma antigen present in the sample, and detecting any complex formed. The biological sample is typically obtained from a human suspected of being afflicted with the carcinoma. Suitable biological samples are serum, blood, sputum, feces, lymph fluid, spinal fluid, lung secretions, and urine, among others. Clearly, any source of fluid, tissue and the like may be prepared for use in this method as is known in the art.

The hybrid BrE-3 analogue peptide of the invention, chimeric BrE-3 polypeptide, and the mouse BrE-3 antibody show substantially no strong binding to normal tissue. The hybrid BrE-3 analogue peptide shows a pattern similar to the BrE-3 chimeric polypeptide and the mouse BrE-3 antibody. The mouse BrE-3 antibody was shown to bind with specificity to carcinoma tumors of the breast, lung, ovary, bladder, and the endometrium, mesothelioma, colon, kidney, liver, merkel cells, pancreas, salivary glands, sarcomas and thyroid, among others. Weak binding was only shown to normal breast tissue, lung tissue, distal convoluted tubes of the kidney, acini of the pancreas and stomach mucosa (Peterson, J. A., et al. (1990), supra). The KC-4 hybrid murine peptide has tissue specificity similar to that of the mouse KC-4 antibody. The KC-4 monoclonal antibody was shown to bind specifically and strongly to solid tumor tissue in the lung, colon, kidney, breast, stomach, prostate, pancreatic, lymph node doctal and lymphoma, and non-specifically and weakly to normal breast, kidney, and stomach tissue. KC-4 also showed some weak binding to normal tissue including spinal cord, uterus, thyroid, tongue, prostate, spin, adrenal, lung, gall bladder, heart, lymph nodes, colon, liver, brain, testes, thymus, and placenta (U.S. Pat. No. 4,708,930). In one preferred embodiment of the in vitro diagnostic method, the anti-carcinoma analogue peptide added to the biological sample comprises a labeled hybrid analogue peptide. Suitable labeling materials were described above. This method may be practiced, with the solid support containing kit described above, as a competitive assay as disclosed by Ceriani, R. L., et al. (Ceriani, R. L., et al. (1 992), supra).

Also provided herein is a method of inhibiting the growth or reducing the size of a primary or metastasized carcinoma comprising administering to a subject in a need of the treatment an effective amount of the anti-carcinoma hybrid analogue peptide of the invention. Typically, the hybrid analogue peptide may be administered in an amount of about 0.001 to 2000 μg/kg body weight per dose, and more preferably about 0.01 to 500 mg/kg body weight per dose. Repeated doses may be administered as prescribed by the treating physician. However, other amounts are also suitable. Generally, the administration of the hybrid analogue peptide is conducted by infusion so that the amount of radiolabel, toxin or other effector agent present that may produce a detrimental effect may be kept under control by varying the rate of administration. Typically, the infusion of one dose may last a few hours. However, also contemplated herein is the constant infusion of a dose for therapeutic purposes that will permit the maintenance of a constant level of the hybrid polypeptide in serum. The infusion of the hybrid analogue peptide of the invention may be conducted as follows. Intravenous (I.V.) tubing may be pretreated, e.g., with 0.9% NaCl and 5% human serum albumin and placed for intravenous administration. The prescribed dose of the analogue peptide may be infused as follows. Unlabeled analogue peptide may be infused initially. 30 minutes after completion of the unlabeled antibody infusion, 111 In-labeled and 90 Y labeled antibody may be co-infused. The I.V. infusion may comprise a total volume of 250 ml of 0.9% NaCl and 5% human serum albumin and be infused over a period of about 2 hours depending on any rate-dependent side effects observed. Vital signs should be taken every, e.g., 15 minutes during the infusion and every one hour post infusion until stable. A thorough cardiopulmonary physical examination may be done prior to, and at the conclusion, of the infusion. Medications including acetaminophen, diphenhydramine, epinephrine, and corticosteroids may be kept at hand for treatment of allergic reactions should they occur. The administration of the hybrid analogue peptide of the invention may be repeated as seen desirable by a practitioner. Typically, once a first dose has been administered and imaging indicates that there could be a reduction in the size of the tumor, whether primary or metastasized, repeated treatments may be administered every about 1 to 100, and more preferably about 2 to 60 days. These repeated treatments may be continued for a period of up to about 2 years, and in some circumstances even for longer periods of time or until complete disappearance of the tumor(s). The administration of the hybrid analogue peptides of this invention is typically more useful for therapeutic purposes when a primary tumor has, for example, been excised. Thus, it is primarily, for mopping up after surgical intervention or in cases of cancerous metastases that the present method is of most use.

Also provided herein is a substantially pure, isolated analogue polydeoxyribonucleotide that comprises an analogue oligodeoxyribonucleotide encoding the analogue peptide or hybrid peptide of this invention, including all redundant sequences. In one preferred embodiment, the analogue polydeoxyribonucleotide of the invention comprises a DNA sequence selected from the group consisting of DNA Sequence ID No: 64 of Table 45, or DNA Sequence ID No: 65 of Table 46, or DNA segments encoding the CDR fragments Sequence ID Nos: 68, 70, 72, 76, 78 or 80 flanked by 1 to 10 murine amino acid sequences, and the N-terminal fragment of 1 to 10 amino acids of murine origin, the remainder amino acids present being of human origin. The above DNA sequences may be cloned for expression under the same promoter.

Also provided herein is a hybrid vector that comprises a vector having the analogue polydeoxyribonucleotide of this invention operatively linked thereto. Typically, vectors capable of replication both in eukaryotic and prokaryotic cells are suitable. When the preparation of a glycosylated analogue polypeptide is desired the vector should be suitable for transfection of eukaryotic host cells. In one preferred embodiment, the hybrid vector comprises the analogue polydeoxyribonucleotide and a polydeoxyribonucleotide comprising an oligodeoxyribonucleotide encoding an effector peptide, the effector peptide-encoding polydeoxyribonucleotide being operatively linked to the vector. As already indicated, the various DNA sequences may be cloned for expression under the same promoter. In addition, the polydeoxyribonucleotide encoding the effector polypeptide may also be cloned for expression under the same promoter.

This invention also encompasses a host cell that has been transfected with the hybrid vector described above. Suitable hosts are prokaryotic and eukaryotic hosts such as bacteria, yeast, and mammalian cells such as insect cells and non-producing hybridoma cells, among others. Suitable vectors and/or plasmids for the transfection of each one of these types of hosts are known in the art and need not be further described herein. Also known in the art are methods for cloning DNA sequences into each one of these types of vectors and for transfecting the different types of host cells. Particularly preferred is the cell line having the ATCC Accesion No. HB 11200 (BrE-3 HZ).

Polyribonucleotides may be obtained by transcription of the polydeoxyribonucloetides described above as is known in the art. Provided herein are analogue polyribonucleotides comprising analogue oligoribonucleotides encoding at least one CDR or an analogue variable region or fragments therof, combinations thereof, and combinations thereof with an effector peptide may be prepared by cloning the desired DNA segments and then transcribing the thus obtained hybrid polydeoxyribonucleotide into the corresponding RNA sequences.

This invention also provides a method of producing an analogue peptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell, or released by the cell, that comprises cloning the analogue polydeoxyribonucleotide of the invention into a vector to form a hybrid vector, transfecting a host cell with the hybrid vector and allowing the expression of the anti-carcinoma analogue peptide, and isolating the anti-carcinoma polypeptide or mixtures thereof. The DNA segment encoding the analogue polypeptide may be obtained by chemical synthesis or by site-specific modification of the sequence encoding the variable region of the xenogeneic species by PCR amplification with specifically designed primers as is known in the art. The fragment DNAs may also be prepared by PCR with primers that introduce a stop codon at a desired position as is known in the art. Preferably, the cloning and transfection steps are conducted by cloning polydeoxyribonucleotides encoding the analogue peptides selected from the group comprising at least one CDR or analogue variable region of the heavy or light chains of the xenogeneic species, antibodies thereof, or fragments therof. The method may further comprise allowing the expressed analogue peptides to interact with one another to form double chain analogue peptides, one or both analogue peptide chain comprising at least one xenogeneic CDR or variable region of the light or heavy chain of the antibody or fragment thereof modified as described above. Still part of this invention is a method of producing a hybrid analogue peptide comprising an effector peptide and an analogue peptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or that is released by the cell, the method comprising transfecting a host cell with the hybrid vector of this invention carrying a DNA sequence encoding the hybrid analogue peptide, allowing the expression of the hybrid analogue peptide, and isolating the hybrid ananlogue peptide or mixtures thereof. The techniques for obtaining mRNA, conducting reverse transcription and PCR amplification of DNA, chemical synthesis of primers, cloning DNA sequences into a vector, transfecting a host cell, and purifying polypeptides from a culture medium are known in the art and need not be further described herein.

This invention also encompasses an anti-idiotype peptide that comprises polyclonal antibodies raised against anti-carcinoma antibodies, the analogue peptide of the invention, monoclonal antibodies thereof, fragments thereof selected from the group consisting of CDRs, Fab, Fab', (Fab')2, and variable region fragments and fragments thereof, analogues thereof selected from the group consisting of Fab, Fab', (Fab')2, and variable regions thereof, wherein about 1 to at least 46 amino acids in the FRs are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in human antibodies, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids. The technique for obtaining anti-idiotype polypeptides is known in the art and need not be further described herein (Nisonoff, A. and Lamoyi, "Implication of the Presence of an Internal Image of an Antigen in Anti-ldiotype Antibodies: Possible Applications to Vaccine Production", Clin. Immunol. Imminopathol. 21:397-406 (1981)). Moreover, the technique for producing hybridomas secreting monoclonal antibodies of a certain specificity is also known in the art (Kohler, G. and Milstein, C. (1975), supra). Techniques for obtaining different antibody fragments were described above or are known in the art and need not be further described herein (Wilbanks, T., et al., "Localization of Mammary Tumors In Vivo with 131 I-Labeled Fab Fragments of Antibodies Against Mouse Mammary Epithelial (MME) Antigens", Cancer 48:1768-1775 (1981)). The techniques for modifying peptides to obtain the analogue peptides of this invention have been described above or are known in the art.

In one particularly preferred embodiment, it is provided a hybrid anti-idiotype polypeptide comprising the anti-idiotype polypeptide of the invention and an effector agent operatively linked to the anti-idiotype polypeptide. Effector agents suitable for use herein were described above for the anti-carcinoma analogue polypeptide of the invention are also suitable for use with the anti-idiotype polypeptide. Preferred are polyclonal antibodies raised against the anti-carcinoma monoclonal antibodies or the analogue peptide of the invention, and a monoclonal antibody obtained by fusion of a B-cell producing an antibody having specificity for the analogue peptide of the invention and an immortalized cell line. Also preferred are fragments of the monoclonal antibody such as Fab, Fab', (Fab')2 and variable region fragments, analogues and fragments thereof as described above, and CDRs. Also, as described above for the anti-carcinoma polypeptide analogue, preferred are combinations of the above fragments and analogues and combinations of the fragments with whole antibodies and analogues thereof. In another preferred embodiment, the anti-idiotype polypeptide comprises an analogue variable region of a monoclonal antibody linked to a peptide comprising the hexamers or trimers described above or tandem repeats thereof.

DNA and RNA segments encoding the anti-idiotype polypeptide and hybrid polypeptidde a hybrid vector having the DNA operatively linked thereto and a host cell transfected with the hybrid vector are also contemplated herein.

Also provided herein is an anti-carcinoma vaccine that comprises the anti-idiotype polypeptide of the invention, and a pharmaceutically-acceptable carrier. Typically, the anti-idiotype polypeptide is present in the composition in an amount of about 0.001 to 99.99 wt %, and more preferably about 0.01 to 50 wt % of the composition. However, other amounts are also suitable. Pharmaceutically-acceptable carriers are known in the art and need not be further described herein. The vaccine provided herein may further comprise other ingredients such as adjuvants, and the like. Examples of adjuvants are SAF-1 and Freund's, among others. Suitably, other ingredients typically used for the preparation of vaccines may also be utilized herein. In one embodiment, the vaccine of the invention may be provided in unit form as a powder or in a diluent. In another embodiment, it may be provided in powder form in a sterile container comprising a plurality of doses for preparation prior to utilization. Diluents that are suitable for the preparation of a formulation that may be administered to a patient by injection are known in the art. Examples were provided above.

An anti-carcinoma vaccination kit is also provided by this invention that comprises, the vaccine described above and a diluent in separate sterile containers, and instructions for its use.

Also provided herein is a method of vaccinating against carcinoma that comprises administering to a human an effective amount of the anti-idiotype polypeptide or hybrid polypeptide described above. Typical amounts administered to a human are about 0.1 to 5000 μg/kg body weight/dose, and more preferably about 1 to 500 μg/kg body weight/dose. The anti-idiotype vaccine of the invention may be administered repeatedly in order to boost the active immunization produced by the first dose. An anti-idiotype antibody very likely resembles the epitope on the carcinoma cell to which the anti-carcinoma antibody binds. Thus, it may be utilized for the production of an immunological response by a subject such as a human or other mammals against its own carcinoma cells.

When an anti-idiotype polypeptide of, e.g., non-human origin is administered to a, e.g., human, it may produce a somewhat detrimental response. Accordingly, in theory, the smaller the non-human amino acid sequence the anti-idiotype polypeptide contains, the lesser the immunogenic response to its xenogeneic sequences it will elicit in a human. Accordingly, preferred anti-idiotype polypeptides are those containing at least one CDR or variable region of a non-human antibody binding specifically to the anti-carcinoma polypeptide described herein, optionally as a hybrid polypeptide. Also preferred are human anti-idiotype antibodies, CDR and variable fragments thereof, and fragments thereof that are operatively linked to an effector agent comprising a human polypeptide that may include the constant region of a human antibody and fragments thereof, non-peptide polymers, monomers and atoms that may be radiolabeled as described above. Other types of constructs are also possible, several of which were described above.

Peptides comprising the sequence APDTRPAPG or fragments thereof comprising hexamers with the trimer TRP or TRP by itself or tandem repeats thereof may also be utilized for clearing from the circulation of a subject molecules such as antibodies and analogue peptides of the invention which have been used for therapeutic purposes. The peptide comprising the hexapeptide or tripeptide sequences may be utilized as a tandem repeat comprising up to about 10,000 repeats of the basic unit, and in some instances up to about 500,000 repeats. In another embodiment, peptides comprising one or more hexapeptides or tripeptides may be operatively linked to other polypeptide sequences of related or unrelated function, which sequences provide bulk that aids the clearance through the liver and/or kidneys of the immunological complex formed between the circulating unbound or residual antibody or polypeptides utilized for the therapy of carcinomas and the hexapeptide. The peptides comprising the hexapeptide or tripeptide may also be provided as a hybrid analogue peptide with other analogue peptides described above. In the absence of such treatment, the therapeutic antibody, which may carry a radioisotope, a toxin or other therapeutic molecules, may remain in the circulation for several days and in some instances weeks. This, in the case of a radioactively labeled antibody or analogue peptide of the invention may produce extensive damage, which is highly detrimental to the health of the patient, and in some instances lethal.

Thus, this invention also provides a method of lowering the serum concentration of a circulating antibody or polypeptide that binds to an antigen on the surface or in the cytoplasm of carcinoma cells or released by the cells comprising administering to a subject the anti-idiotype polypeptide described above, in an amount effective to bind the circulating polypeptide, to thereby accelerate its clearance. In one preferred embodiment a tandem repeat of up to about 20,000 and even up to about 50,000 peptides comprising the sequence APDTRPAPG, hexamers thereof having a TRP trimer or tandem repeats thereof are preferred. Another preferred embodiment comprises an oligopeptide comprising one or more of the hexamer or trimer sequences and having a molecular weight of about 5,000 to 1,000,000. Typically, the anti-idiotype polypeptide is administered in an amount of about 0.01 to 5000.00 μg/kg body weight/dose, and more preferably about 1 to 250 μg/kg body weight/dose. However, other amounts may also be utilized. The administration of the anti-idiotype polypeptide may be infusion as described above.

Also provided herein is a method of inhibiting the growth or reducing the size of a primary or metastasized carcinoma tumor comprising administering to a subject in need of the treatment an effective amount of an antibody or an anti-carcinoma hybrid analogue peptide comprising an effector agent selected from the group consisting of radioisotopes, therapeutic drugs and vaccines, and an anti-carcinoma polypeptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or released by the cell, allowing the hybrid polypeptide to reach the tumor and the polypeptide to bind thereto, and administering to the subject an amount of the anti-idiotype polypeptide of the invention effective to bind residual or unbound circulating hybrid analogue peptide to thereby accelerate the clearance of the hybrid polypeptide.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless so specified.

EXAMPLES Example 1

Methods Utilized

The procedures utilized herein for the reverse-transcription (RT) of RNAs encoding the variable regions and the subsequent amplification of the cDNAs by the polymerase chain reaction (PCR) have been described (Orlandi, R., et al., "Cloning Immunoglobulin Variable Domains for Expression by the Polymerase Chain Reaction", P.N.A.S. (USA) 86:3833-3837 (1989); Coloma, M. J., et al., "Primer Design for the Cloning of Immunoglobulin Heavy-Chain Leader-Fvs from Mouse Hybridoma Cells Using the PCR", Bio.Techniques 11:152-156 (1991); Gavilondo-Cowley, J. V., et al., "Specific Amplification of Rearranged Immunoglobulin Fv Genes from Mouse Hybridoma Cells", Hybridoma 9:407-417 (1990)).

Total RNA is an adequate substrate for RT-PCR. Polyadenylated RNA was utilized herein, however, because it contains only minor levels of contaminating ribosomal RNA and practically no DNA. The polyadenylated RNA was isolated with a Fast Track mRNA isolation kit (Invitrogen Corporation, San Diego, Calif.).

The oligonucleotides were synthesized on a PCR-Mate EP DNA synthesizer model 391 (Applied Biosystems, Foster City, Calif.). A PCR mouse Ig primer set was purchased from Novagen (Madison, Wis.), and complementary DNA (cDNA) was prepared with an RNA PCR kit (Perkin Elmer-Cetus, Norwalk, Conn.).

PCR DNA fragments were cloned directly into pCR1000, using a TA cloning kit (Invitrogen Corporation, San Diego, Calif.). Plasmid DNA was isolated with a kit purchased from Qiagen (Tchapsworth, Calif.), and DNA sequencing was conducted with a Sequenase 2.0 DNA sequencing kit (United States Biochemical, Cleveland, Ohio) using aqueous 5'α-35 SdATP at 600 mCi/mmol (Amersham Corporation, Arlington Heights, Ill.).

Sequence analyses were performed on a Macintosh computer using the program GeneWorks (IntelliGenetics, Inc, Mountain View, Calif.).

Example 2

Tissue Culture Media

SP2/0-Ag14 cells (Shulman, M., et al. (1978), below) were cultured either in Dulbecco's modified Eagle's medium (DME): fetal bovine serum (FBS), 90:10 (v/v) or in a mixture of DME:RPMI:FBS, 45:45:10 (v/v/v) or RPMI:FBS, 90:10 (v/v). Penicillin and streptomycin were added to prevent bacterial growth. When serum-free medium was utilized, it contained an HL-1 supplement as directed by the manufacturer (Ventrex Labs., Portland, Me.). The freezing medium was 10% DMSO in bovine serum.

Example 3

PCR Primers

Primers and primer mixtures MulgκVL 5'-C, MulgλVL 3'-1, MulgVH 5'-C, MulgVH 5'-F, and MulgλVH 3'-2 were part of a primer set purchased from Novagen. Their sequences may be obtained from Novagen. Other primers were synthesized by the inventors. These sequences are shown in Table 12 below.

                                  TABLE 12__________________________________________________________________________Synthetic Primers__________________________________________________________________________JO2- T GAA GCT TGC TCA CTG GAT GGT GGG AA (Seq. ID No: 1);JO3- AGA TGG GGG TGT CGT TTT GG (Seq. ID No: 2);JO4- GCT TGA ATT CCA GGG GCC AGT GGA TAG A (Seq. ID No: 3);VH 1BACK (4) - AG GT(CG) (CA)A(GA) CTG CAG (CG)AG TC(TA) GG(Seq. ID No: 4)JO14- ATG TAC TTG GGA CTG AAC TAT GTC TT (Seq. ID No: 5).__________________________________________________________________________ *Orlandi, R., et al. (Orlandi, R., et al. "Cloning Immunoglobulin Variabl Domains for Expression by the Polymerase Chain Reaction", P.N.A.S. (USA) 86: 3833-3837 (1989)).
Example 4

Cloning of Chimeric BrE-3 Antibody Polydeoxyribonucleotide

Two expression vectors pAG4622 and pAH4604 were utilized herein (Coloma, M. J., et al., "Novel Vectors for the Expression of Antibody Molecules Using Variable Regions Generated by PCR", J. Immunol. Methods 152:89-104 (1992)). These were kindly provided by S. L. Morrison (Dept. of Microbiology and Molecular Genetics, UCLA). The construction and expression of chimeric genes was performed as described by Coloma, M. J., et al. (Coloma, M. J., et al. (1992), supra).

Oligonucleotides were synthesized and used in a PCR mixture to produce variable heavy (VH) and variable light (VL) fragments with the correct ends for insertion into the pAG4622 and pAH4604 expression vectors. There sequences are shown in Table 13 below.

                                  TABLE 13__________________________________________________________________________Synthesized Oligonucleotides__________________________________________________________________________JO16 (sense VH leader) - GGG GATATC CACC ATG TAC TTG GGA CTG AACTAT GTC UC A (Seq. ID No: 6);JO17 (sense VL leader) - GGG GATATC CACC ATG AAG TTG CCT GTT AGGCTG TTG GT (Seq. ID No: 7);JO18 (anti-sense JH3) - GGG GCTAGC TGC AGA GAC AGT GAC CAG AGT CC(Seq. ID No: 8);JO19 (anti-sense Jκ1) - GGG GTCGACTTAC G TTT GAT TTC CAG CTT GGTGCC TCC A (Seq. ID No: 9).__________________________________________________________________________

The original pCR1000 clones were utilized as the starting templates for the PCR. The new PCR products were cloned back into pCR1000 and their sequence confirmed. Correctly modified and amplified fragments were excised with either EcoRV and Sal I (for VL) or with EcoR V and Nhe I (for VH). These fragments were then ligated into the respective vectors, which had been cut open with the appropriate restriction enzymes. Both the vectors and the inserts were purified from an agarose gel prior to ligation, using the Bio101 GeneClean kit (glass beads) (La Jolla, Calif.).

Example 5

Expression of Mouse-Human Chimeric Antibody

The VH and VL regions in the final mouse-human chimeric antibody were sequenced once again to verify that their sequences were correct.

The non-producer myeloma cell line SP2/0-Ag14, (ATCC: CRL 1581, Shulman, M., et al., "A Better Cell Line for Making Hybridomas Secreting Specific Antibodies", Nature 276:269-270, (1978)) was transfected, and a chimeric antibody were isolated as described by Coloma, M. J. et al. (1992), with the following modification. The selection was only undertaken for the uptake of hisD by adding 5 mM histidinol to the medium and readjusting the pH to 7.4 with NaOH.

Example 6

Production of Transfected Hosts

After ten days, the stable transfectant colonies were clearly established at a frequency of approximately 10-5. The colonies were transferred to a normal medium (without histidinol) and the supernatants from stable transfectants were assayed for the presence of the mouse-human chimeric BrE-3 antibody. This was done by capturing the secreted mouse-human chimeric BrE-3 antibody with a plate-bound goat anti-human-κ antibody and developing with goat anti-human-γ antibody as described by Coloma, M. J. et al. with the following modification. The secondary antibody utilized herein was radiolabeled with 125 I.

Example 7

Confirmation of Mouse-Human Chimeric BrE-3 Antibody Expression

The supernatants were assayed for binding to human milk fat globule (HMFG) as described by Ceriani R. L. (Ceriani R. L., et al., Diagnostic Ability of Different Human Milk Fat Globule Antigens in Breast Cancer", Breast Cancer Res. Treat. 15:161-174 (1990)). HMFG was bound to the microtiter plates as described previously (Ceriani R. L., "Solid Phase Identification and Molecular Weight Determination of Cell Membrane Antigens with Monoclonal Antibodies", in: Monoclonal antibodies and functional cell lines. Progress and application, Bechtol, K. B., McKern, T. J., and Kennett, R., Eds., Plenum Press, New York, pp 398-402 (1984)).

Most colony supernatants were positive by both assays. The colonies that secreted the highest level of chimeric antibody into the supernatants, as determined by these assays, were subcloned and subsequently adapted to serum-free medium for the purification of antibody.

Example 8

Competition Assay

The antibody-antigen affinity constants for the mouse-human chimeric antibody which binds to human milk mucin and the whole murine antibody were determined by obtaining the reciprocal value of the concentration of competing unlabeled monoclonal antibody giving 50% binding as described by Sheldon, K. et al. (Sheldon, K.,et al., "Characterization of Binding of Four Monoclonal Antibodies to the Human Ovarian Adenocarcinoma Cell Line HEY", Biochem. Cell Biol., 65: 423-428, (1987)). The protocol for the assay was as follows.

Microtiter plates (Dynatech, Chantilly, Va.) were prepared using successive layers of methylated BSA, glutaraldehyde, anti-β-galactosidase and the bacterial fusion protein 11-2 (a hybrid of β-galactosidase and human mammary mucin) as described in Ceriani, R. L., et al. (Ceriani, R. L., et al., "A Novel Serum Assay for Breast Cancer Epithelial Antigen Using a Fusion Protein", Anal. Biochem. 201:178-184 (1992). Each well contained 388 ng of the 11-2 fusion protein. To each well were added 25 μl 125 I -BrE-3 (ATCC No. HB 10028) in RIA buffer (10% bovine calf serum, 0.3% triton X-100, 0.05% sodium azide pH7.4, in phosphate buffer saline), and competed with 25 μl of either unlabeled murine antibody or mouse-human chimeric antibody in RIA buffer at final concentrations in the nanomolar range.

Iodinations were performed with 125 I (17 Ci/mg, Nordion International Inc., Kanata, Ontario, Canada). 50 micrograms of monoclonal antibody BrE-3 (Coulter, Hialeah, Fla.) were labeled at a specific activity of 9.56 mCi/mg using the chloramine T method as described by Ceriani, R. L. and Blank, E. W., (Ceriani, R. L., and Blank, E. W., "Experimental Therapy of Human Breast Tumors with 131I-Labeled Monoclonal Antibodies Prepared Against the Human Milk Fat Globule", Cancer Res. 48:4664-4672 (1988)).

When the counts of bound radiolabeled murine BrE-3 antibody were plotted an the Y axis and the logarithm of the nanomolar (nM) concentration of competing unlabeled murine BrE-3 antibody or mouse-human chimeric antibody were plotted in the X axis, both curves overlapped within 5% error (Figure not shown).

This proves that the variable region's affinity characteristics have been preserved.

Example 9

Amplification of cDNAs Encoding BrE-3 Variable Regions

The cDNAs that encode the BrE-3 mouse immunoglobulin variable domains (VH and VL) were prepared by reverse transcription and PCR amplification (RT-PCR) from polyadenylated RNA isolated from 108 BrE-3 hybridoma cells by the following procedure.

The JO2, JO3, JO4, JO14 and VH 1BACK primers were synthesized, and there sequences shown in Example 3 above. Other primers were purchased from Novagen. With the exception of VH 1BACK, which is a framework-specific primer, all sense primers are specific for the leader peptide region. All anti-sense primers are specific for the constant regions. The degenerate λ chain of the specific primer MulgλVL 3'-1 (from Novagen), was used to isolate the κ chain cDNA clones because of the similarity of the γ and κ. An identical κ chain clone was isolated with primer JO2 which is specific for the κ chain constant domain.

The VH region cDNA could not be isolated with the available leader peptide primers. Thus, the VH 1BACK primer was used, which yielded the VH cDNA γ72. The leader-peptide primer JO14 was then designed by extrapolating from the framework sequence of γ72, using cataloged nucleotide sequences (Kabat, E. A., et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, NIH publication No. 91-3242, 5th Edition (1991). After sequential PCR reactions, this new primer yielded the complete VH framework cDNA. This information is summarized in Table 14 below.

              TABLE 14______________________________________Primer Combinations for PCR AmplificationClone No.     Sense Primers                      Antisense Primers______________________________________VL  152        MulgκVL5'-C                          JO2  164        MulgκVL5'-C                          MulgλVL3'-1VH  γ72  VH 1BACK                          (JO3 or JO4)  1012       JO14 (1st PCR)                          JO3             JO14 (2nd PCR)                          JO4  1043       JO14 (1st PCR)                          JO3             (MulgVH5'-C +                          MulgγVH3'-2             MulgVH5'-F)             (2nd PCR)______________________________________
Example 10

Isolation of Amplified BrE-3 VL and VH cDNA and Sequences

The PCR products were cloned without prior purification into pCR1000 (Invitrogen) and sequenced in both directions. Clones 152, 164, 1012, and 1043 were isolated independently during different RT-PCR runs. The sequences of VL clones 152 and 164 were found to be identical, as were the sequences of the VH clones 1012, 1043. The VH and VL DNA sequences and their derived protein sequences are shown in Tables 15 and 16 below.

                                  TABLE 15__________________________________________________________________________BrE-3 VL Nucleotide and Derived Protein Sequences__________________________________________________________________________BrE-3 VLDNA SequenceATG   AAG TTG    CCT       GTT          AGG             CTG                TTG                   GTG                      CTG                         TTG                            TTC                               TGG                                  ATT                                     CCT                                        GCT                                           TCC                                              ATC                                                 (Seq. ID No: 10)AGT   GAT GTT    GTG       ATG          ACC             CAA                ACT                   CCA                      CTC                         TCC                            CTG                               CCT                                  GTC                                     AGT                                        CTT                                           GGA                                              GAT                                                 CAAGCT   TCC ATC    TCT       TGC          AGA             TCT                AGT                   CAG                      AAC                         CTT                            GTA                               CAC                                  AAC                                     AAT                                        GGA                                           AAC                                              ACC                                                 TATTTA   TAT TGG    TTC       CTG          CAG             AAG                TCA                   GGC                      CAG                         TCT                            CCA                               AAG                                  CTC                                     CTG                                        ATT                                           TAT                                              AGG                                                 GCTTCC   ATC CGA    TTT       TCT          GGG             GTC                CCA                   GAC                      AGG                         TTC                            AGT                               GGC                                  AGT                                     GGA                                        TCA                                           GAG                                              ACA                                                 GATTTC   ACA CTC    AAG       ATC          AGC             AGA                GTG                   GAG                      GCT                         GAG                            GAT                               CTG                                  GGA                                     GTT                                        TAT                                           TTC                                              TGC                                                 TTTCAA   GGT ACA    CAT       GTT          CCG             TGG                ACG                   TTC                      GGT                         GGA                            GGC                               ACC                                  AAG                                     CTG                                        GAA                                           ATC                                              AAA                                                 CAmino Acid Sequencem k l p v r I I v I L F W I P A S I S D1 V V M T Q T P L S L                                                 (Seq. No: 11)P V S L G D Q A S I S C R S S Q N L V H N N G N T Y L Y W FL Q K S G Q S P K L L I Y R A S I R F S G V P D R F S G S GS E T D F T L K I S R V E A E D L G V Y F C F Q G T H V P WT F G G G T K L E I K__________________________________________________________________________

                                  TABLE 16__________________________________________________________________________BrE-3 VH Nucleotide and Derived Protein Sequences__________________________________________________________________________BrE-3 VHDNA SequenceATG   TAC TTG    GGA       CTG          AAC             TAT                GTC                   TTC                      ATA                         GTT                            TTT                               CTC                                  TTA                                     AAA                                        GGT                                           GTC                                              CAG                                                 (Seq. ID No: 12)AGT   GAA GTG    AAG       CTT          GAG             GAG                TCT                   GGA                      GGA                         GGC                            TTG                               GTG                                  CAA                                     CCT                                        GGA                                           GGA                                              TCC                                                 ATG                                                    AAA                                                       CTCTCT   TGT GCT    GCT       TCT          GGA             TTC                ACT                   TTT                      AGT                         GAT                            GCC                               TGG                                  ATG                                     GAC                                        TGG                                           GTC                                              CGC                                                 CAG    TCT                                                 CCAGAG   AAG GGG    CTT       GAG          TGG             GTT                GCT                   GAA                      ATT                         AGA                            AAC                               AAA                                  GCC                                     AAT                                        AAT                                           CAT                                              GCA                                                 ACA    TAT                                                 TATGAT   GAG TCT    GTG       AAA          GGG             AGG                TTC                   ACC                      ATC                         TCA                            AGA                               GAT                                  GAT                                     TCC                                        AAA                                           AGT                                              AGA                                                 GTG    TAC                                                 CTGCAA   ATG ATA    AGC       TTA          AGA             GCT                GAA                   GAC                      ACT                         GGC                            CTT                               TAT                                  TAC                                     TGT                                        ACT                                           GGG                                              GAG                                                 TTT    GCT                                                 AACTGG   GGC CAG    GGG       ACT          CTG             GTC                ACT                   GTC                      TCT                         GCA                            GAmino Acid Sequencem y l g t n y v f l V F L L K G V Q S E1 V K L E E S G G G L                                                 (Seq. ID No: 13)V Q P G G S M K L S C A A S G F T F S D A W M D W V R Q S PE K G L E W V A E I R N K A N N H A T Y Y D E S V K G R F TI S R D D S K S R V Y L O M I S L R A E D T G L Y Y C T G EF A N W G Q G T L V T V S A__________________________________________________________________________

The sequences were interpreted as described by Kabat et al. (1991). The residues that are shown in lower case correspond to PCR primers. The mature chains begin at D1 (VL) and E1 (VH), respectively. The amino-acids that are underlined are those corresponding to the CDRs. The underlined nucleotides indicate joining segments.

The framework and CDR polypeptide segments were identified according to Kabat et al. (1991). VL is a group IIκ chain. Part of the CDR 3 and all of framework 4 (FR4) are encoded by Jk1. VH belongs to group IIIc. CDR 3 and FR4 are encoded by JH3. Little or nothing remains from an unidentified D minigene. Thus, the CDR 3 is only 4 amino-acids long.

Example 11

Comparison of cDNA deduced Amino Acid Sequence with Directly Determined N-Terminal Fragment Sequence

Table 17 below shows a comparison between the cDNA-derived polypeptide sequence and the polypeptide sequence determined directly from purified BrE-3 monoclonal antibody.

              TABLE 17______________________________________Comparison of cDNA-Deduced Protein Sequence withDirectly Determined N-terminal Protein SequenceVL    cDNA-deduced             DVVMTQTPLSLPVSLGDQASISCRSVL    Protein sequence             GVVMTQTPLSLPVVLGDQASIIXRXVH    cDNA-deduced             EVKLEESGGGLVQPGGSMKLSCAASVH    Protein sequence             EVKLEESGGVLVQPGGSMKLSSAAS______________________________________

BrE-3 was reduced with 5% mercaptoethanol, separated on a 10% SDS polyacrylamide gel, and electroblotted onto a ProBlott membrane (Applied Biosystems, Foster City, Calif.). Amino acid sequencing was performed directly on the immobilized bands by the Biotechnology Instrumentation Facility, University of California, Riverside. The protein sequence given here is the sequencer's best guess.

Once the variable region cDNAs were cloned, it was confirmed that, in fact, they encoded the variable regions of BrE-3 and not those of another antibody by comparing the cDNA-derived amino acid sequences of the cloned BrE-3 variable region with the N-terminal sequence of purified BrE-3 antibody directly determined by a single run of protein sequencing. The cDNA sequences were shown to be accurate by comparison with 2 independently reverse transcribed clones.

The general agreement between the predicted and the determined amino-acid sequences shows that the cloned cDNAs encode polypeptides of the same class and subclass as the variable regions of BrE-3. This indicates that the cDNAs encode authentic variable regions. The authenticity of the variable region polypetide and, therefore, that of the mouse-human chimeric BrE-3 antibody is unquestionable given that the variable regions and the chimeric antibody affinity constant is indistinguishable from that of BrE-3.

Example 12

Construction of Mouse-Human Chimeric Antibody Genes

The vectors used were developed by Coloma, M. J., et al. (Coloma, M. J., et al. (1992), supra) and kindly provided by S. L. Morrison (Dept. of Microbiology and Molecular Genetics, UCLA). Both vectors were derived from pSV2 (Mulligan, R. C., and Berg, P., "Expression of a Bacterial Gene in Mammalian Cells", Science 209:1422-1427 (1980)), and contain genomic fragments encoding either the heavy or the light chain constant domains. The vectors accept cDNAs that encode the Fv regions. To ligate the Fv cDNAs to the vectors, restriction ends were added to the cDNAs in a set of PCR reactions, using-the JO16, JO17, JO18 and JO19 primers.

The pAG4622 light chain vector contains the gene for the human κ chain constant region, including the J-C intron. It encodes xanthine-guanine phosphoribosyltransferase or gpt (Mulligan, R. C., and Berg, P., "Selection for Animal Cells that Express the Escherichia Coli Gene Coding for Xanthine-Guanine Phosphoribosyltransferase",P.N.A.S. (USA) 78:2072-2076 (1981)) as a dominant selectable marker. It accepts the mouse VL cDNA between the ribosome binding site (Kozak, M., "Compilation and Analysis of Sequences Upstream from the Translational Start Site in Eukaryotic mRNAs", Nucleic Acids Res. 12:857-872 (1984)), which is preceded by the VH promoter from the anti-dansyl murine monoclonal antibody 27.44 (Coloma, M. J., (1992), supra), and the J-C intron. The J-C intron contains the κ chain enhancer (Potter, H., et al., "Enhancer-Dependent Expression of Human κ Immunoglobulin Genes Introduced into Mouse Prep-B Lymphocytes by Electroporation", P.N.A.S. (USA) 81:7161-7165 (1984); Emorine, L., et al., "A Conserved Sequence in the Immunoglobulin J Kappa-C Kappa Intron: Possible Enhancer Element", Nature 304: 447-449 (1983)).

The pAH4604 heavy chain vector contains the gene for the heavy chain γ1 constant region, but no J-C intron. It encodes histidinol-dehydrogenase or hisD (Hartman, S. C. and Mulligan, R. C. Two Dominant-Acting Selectable Markers for Gene Transfer Studies in Mammalian Cells", P.N.A.S. (USA) 85:8047-8051 (1988)) as a dominant selectable marker. It accepts the mouse VH cDNA between the dansyl promoter-ribosome binding site and the constant γ1 gene. The vector also contains an insert that encodes the heavy chain enhancer (Rabbitts, T. H., et al, "Transcription Enhancer Identified Near the Human C mu Immunoglobulin Heavy Chain Gene is Unavailable to the Translocated c-myc Gene in a Burkitt Lymphoma", Nature 306:806-809 (1983)).

The new VH and VL DNA fragments with appropriate restriction ends were integrated into pAH4604 and pAG4622 as described in Example 4 above. The vectors were then electroporated (together) into SP2/0-Ag14 myeloma cells as described by Coloma et al. (1992), supra.

Example 13

Characterization of Mouse-Human Chimeric BrE-3 Antibody and VH and VL Regions

The supernatants from stable transfectants were assayed for the presence of the mouse-human chimeric antibody as described in Examples 6 and 7 above. High producing transfectants were subcloned and subsequently adapted to grow in serum-free medium. The mouse-human chimeric antibody produced by the myeloma cell line was then purified from the culture supernatant using a Sepharose 4B-protein A column (Bio-Rad, Richmond, Calif.) as described in Ey, P. L., et al. (Ey, P. L., et al., "Isolation of Pure IgG1, IgG2a and IgG2b Immunoglobulins from Mouse Serum Using Protein A-Sepharose", Immunochemistry 15:429-436 (1978)). Antibody disulfide bonds were reduced to separate the light and heavy chains by heating for 10 min at 65° in Laemmli loading buffer containing 5% beta-mercaptoethanol. The separated chains were then chromatographed on a SDS polyacrylamide gel (10%). The reduced mouse-human chimeric antibody and BrE-3 antibody were eletrophoresed in separate lanes next to 97.4, 66.2, 45.0, 31.0 and 2.5 Kdalton protein markers. Table 18 below shows the apparent molecular weights of the two bands obtained for both.

              TABLE 18______________________________________Reduced BrE-3 and Chimeric BrE-3 AntibodyVL and VH Apparent Molecular WeightsChimeric Antibody      BrE-3______________________________________VH  VL       VH                         VH(Kd)     (Kd)          (Kd)   (Kd)50       30            49     29______________________________________

The heavy and light chains of the chimeric BrE-3 antibody separate as expected when electrophoresed on a polyacrylamide gel.

Example 14

Affinity Binding Constants for BrE-3 and Mouse-Human Chimeric Antibody

The purified mouse-human chimeric BrE-3 antibody and purified murine BrE-3 gave similar competition curves when tested against 125 I-labeled murine BrE-3 binding to its antigen. The affinity binding constants of the murine antibody and the mouse-human chimeric antibody were determined in independent competition assays as described in Example 8 above. The values of the constants are 2.68×108 M-1 and 3.75×108 M-1 for the hybrid BrE-3 polypeptides and for the murine antibody of BrE-3, respectively. These values are not distinguishable at a 95% confidence interval.

Example 15

Tissue Binding Studies

Immunohistochemical staining using the immunoperoxidase technique of consecutive human breast carcinoma tissue sections was conducted with the mouse-human chimeric BrE-3 antibody. A control was stained with the anti-human secondary antibody only. Positive staining resulted from the use of the mouse-human chimeric BrE-3 antibody, followed by the anti-human antibody specific binding. (Pictures not shown).

The breast carcinoma tissue sections were stained with the supernatant of the transfected cells using the Vectastain ABC method (Vector Labs, Burlingame, Calif.). The tissue stained with the goat anti-human Ig secondary antibody only shows background or non-specific staining of necrotic areas of the tissue section.

The tissue stained with mouse-human chimeric BrE-3 antibody, followed by the secondary antibody, shows specific staining of the breast carcinoma cells in the breast tissue sections.

Example 16

BrE-3 Imaging Studies

The murine monoclonal antibody BrE-3 has been shown to be highly effective for imaging and for the radioimmunotherapy of breast cancers. For example, in a pharmacokinetic study of 15 breast cancer patients conducted with an 111 In MXDTPA-BrE-3 radioimmunoconjugate (BrE-3 antibody), the serum levels were low in most patients, the blood clearance correlated with the circulating antigen and the imaging results showed that about 86% of all sites could be imaged (Liebes, L., et al., "Pharmacokinetics of 111 In-BrE-3 Monoclonal Antibody in Patients with Breast Carcinoma", Proc. Am. Assoc. Cancer Res. 33:216(Abs No. 1292) (1992)).

A 90 Y-BrE-3 radioimmunoconjugate having similar pharmacokinetic characteristics and extrapolating the 111 In-BrE-3 dosimetry results provide a superior therapeutic agent, as well.

As with many other monoclonal antibodies, however, the clinical applications of BrE-3, a whole mouse antibody, are limited by the HAMA response. A chimeric monoclonal antibody should give a more restricted HAMA response.

Example 17

Hybrid BrE-3 Immunogenicity

The BrE-3 variable region polypeptides have been cloned without the constant regions to produce less immunogenic polypeptides than the parent murine antibody. It has, moreover, been shown herein that the mouse-human chimeric BrE-3 antibody lacking its original murine constant region preserves its antigen binding characteristics.

A BrE-3 variable region chimeric alone or as a mouse-human chimeric antibody also containing a constant region human region or a fragment thereof is significantly less immunogenic to humans than the parent murine antibody. The hybrid polypeptide comprising the variable region of the BrE-3 antibody and the constant region of a human antibody was shown to preserve the original binding affinity of the murine antibody.

In this hybrid polypeptide, approximately 2/3 of its contiguous non-human immunogenic targets (CL and CH regions) were entirely replaced by human constant domains.

Example 18

Cloning of KC-4 VH and VL and cDNAs

The procedure and media employed are described and referenced in Examples 1 and 2 above.

Example 19

PCR Primers used in First Isolation of KC-4 cDNAs

The PCR primers were purchased from Novagen (Madison, Wis.). Their sequences, reproduced from the booklet provided by Novagen, are shown in Table 19 below.

                                  TABLE 19__________________________________________________________________________PCR Primer Sequences__________________________________________________________________________MulgκVL 5'-C: sense primer mix for kappa leader.ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTG (Seq. ID No: 14)ACTAGTCGACATGGAGWCAGACACACTCCTGYTATGGGT (Seq. ID No: 15)ACTAGTCGACATGGATTTWCAGGTGCAGATTWTCAGCTTC (Seq. ID No: 16)MulgκVL 3'-1: antisense kappa constant region.CCCAAGCTTACTGGATGGTGGGAAGATGGA (Seq. ID No: 17)MulgVH 5'-F: sense primer mix for heavy chain leader.ACTAGTCGACATGRACTTTGGGYTCAGCTTGRTTT (Seq. ID No: 18)ACTAGTCGACATGAGAGTGCTGATTFCTTTTGTG (Seq. ID No: 19)ACTAGTCGACATGGATTTTGGGCTGATTTTTTTTATTG (Seq. ID No: 20)MulgγVH 3'-2: antisense gamma constant region.CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG (Seq. ID No: 21)__________________________________________________________________________
Example 20

Cloning of Mouse-Human Chimeric KC-4 Antibody Ribonucleotide

The two expression vectors pAG4622 and pAH4604 described in Example 4 were utilized.

Oligonucleotides synthesized and used in a PCR to produce VH and VL fragments with the correct ends for insertion into the pAG4622 and pAH4604 expression vectors are shown in Table 20 below.

                                  TABLE 20__________________________________________________________________________PCR Primers Sequences__________________________________________________________________________JO20 - sense kappa leaderGGG GATATC CACC ATG AAG TTG CCT GTT AGG CTG TTG (Seq. ID No: 22)JO21 - antisense JK2CCC GTCGACTTAC G TTT TAT TTC CAG CTT GGT CCC CCC T (Seq. ID No: 23)JO22 - sense VH leaderGGG GATATC CACC ATG GAC TTT GGG CTC AGC TTG GTT TT (Seq. ID No: 24)JO24 - antisense JH3CCC GCTAGC TGC AGA GAC AGA GAC CAG AGT CC (Seq. ID No: 25)__________________________________________________________________________

The original pCR1000 clones were the starting templates for the PCR and the rest of the procedures as described in Example 4 above.

Example 21

Expression of the KC-4 Chimeric Gene

The VH and VL regions in the KC-4 mouse-human chimeric antibody were sequenced once again to verify that their sequences were correct. The transfection of the non-producer myeloma cell line SP2/0-Ag14, (ATCC: CRL 1581) and isolation of polypeptide was conducted as described in Example 5 above.

Example 22

Production of Transfected Hosts

After ten days, stable transfectant colonies were clearly established at a frequency of approximately 1/10,000. The colonies were transferred to normal medium and the assays conducted as described in Example 6 above.

Example 23

Confirmation of Mouse-Human Chimeric KC-4 Antibody Expression

The supernatants were essayed for binding to human milk fat globule (HMFG) and the breast epithelial mucin (BEM) as described previously in Example 7 above. HMFG and BEM were bound to the microtiter plates as described previously by Ceriani, R. L. (1990). In this radioassay the bound chimeric KC-4 (HMFG and BEM) was detected by anti-human gamma chain conjugated to 125- I. Most colony supernatants were positive by both assays. The colonies that secreted the highest level of chimeric antibody in the supernatants, as determined by these assays, were subcloned.

Example 24

Western Blot

75 μl of the culture supernatant was added to 20 μl of 4×Laemmli buffer and 5 μl β-mercaptoethanol and the mixture was heated at 65° C. for 15 min., in order to reduce antibody disulfide bonds and, thus, separate heavy from light chains. 20 μl of the treated sample was chromatographed in duplicate lanes on a 10% SDS polyacrylamide gel together with other antibodies that were treated similarly and that were loaded for comparison. Pre-stained size markers (BioRad, Richmond, Calif.) were also loaded.

The chromatographed proteins were electroblotted onto a ProBlott membrane (Applied Biosystems, Foster City, Calif.) in 90% 30 mM CAPS pH11, 10% methanol, for 1 hour at 25 V and at 4° C. The membrane was cut into 2 parts containing identical antibody samples. The 2 membranes were immersed in 20% Bovine Calf Serum in PBS and shaken slowly at room temperature for 1hour 35 min. 125 I labeled goat anti-human κ chain antibody was added to one membrane and 125 I labeled goat anti-human γ chain antibody to the other membrane. Antibodies were labeled at a specific activity of approximately 10 mCi/mg using the chloramine T method as described by Ceriani, R. L. and Blank, E. W. (1988), the labeled antibodies were diluted to 4,000 cpm/μl in RIA buffer.

After incubating 3 hours at room temperature the blots were washed twice in TBS for 10 min each time, once in TBST (50 mM TRIS pH7.5, 3 mM EDTA 25 mM NaCl) 10 min and once more in TBS (TBS with 0.05% Tween 20) for 10 min. The membranes were dried and exposed to Kodak XAR film.

Western blot analyses of culture supernatants revealed that 3 antibody chains were expressed that corresponded to the 3 antibody chains seen in the original KC-4 antibody. These were a heavy chain that stained with goat anti-human γ chain 125 I-labeled antibody, and 2 light chains that stained with goat anti-human κ chain 125 I-labeled antibody (Figure not shown).

The treatment of the original murine KC-4 antibody with N-glycosidase F (Boehringer Mannheim GmbH Germany) following the recommendations of the manufacturer, produced a noticeable decrease in the intensity of the "top" light chain and a concomitant increase in the intensity of the bottom light chain (Figure not shown).

The explanation for the existence of an extra light chain is that this chain is glycosylated. Three lines of evidence substantiate this. First, the detection of an asparagine-linked glycosylation site in the amino acid sequence of the light chain. That is the triad NIS (Asn-IIe-Ser) in framework 3. Second, the decrease of the intensity in the putative glycosylated band after treatment with N-glycosidase F, while concomitantly the intensity of the non-glycosylated band was increased. Finally, 2 corresponding light chain bands are seen in the chimeric antibody version.

The extra light chain in the chimeric version cannot be a contaminant since it was specifically stained by goat anti-human κ chain antibody. It can only be a product expressed by pAG4622. Thus both light chains must have the same VL amino acid sequence and the same human constant region. These observations show that approximately half of the light chains of both the murine KC-4 and the KC-4 chimeric antibody are glycosylated at the asparagine-linked glycosylation site.

Example 25

Amplification of cDNAs Encoding KC-4 Fv Regions

The cDNAs that encode the KC-4 mouse immunoglobulin VH and VL were prepared as described in Example 9 above from polyadenylated RNA isolated from 100 million KC-4 hybridoma cells. All clones were obtained from independent PCRs. The sequences of the primers are given in Example 19 and 20 above. All primers are specific for either the leader peptide region or for the constant regions. The primer combinations utilized herein are shown in Table 21 below.

              TABLE 21______________________________________Primer Combination for PCR Amplifications   Clone No.            Primer combinations______________________________________VL   96         MulgκVL 5'-C + MulgκVL 3'-1     107        MulgκVL 5'-C + MulgκV3L 3'-1     K1         JO20 + JO21VH   66         MulgVH 5'-F + MulgγVH 3'-2     209        MulgVH 5'-F + MulgγVH 3'-2     H3         JO22 + JO24     H7         JO22 + JO24______________________________________
Example 26

Isolation of Amplified KC-4 FVL (VL) and FVH (VH) cDNA and sequences

The PCR products were cloned, without prior purification, into pCR1000 (Invitrogen) and sequenced in both directions. The VH and VL DNA sequences and their derived protein sequences are shown in Tables 22 and 23 below.

                                  TABLE 22__________________________________________________________________________VL Nucleotide SequencesKC-4 VL (kII-Jk2)__________________________________________________________________________ATG   AAG TTG    CCT       GTT          AGG             CTG                TTG                   GTG                      CTG                         ATG                            TTC                               TGG                                  ATT                                     CCTGCT   TCC AGC    AGT       GAT          GTT             TTG                ATG                   ACC                      CAA                         ACT                            CCT                               CTC                                  TCC                                     CTGCCT   GTC AGT    CTT       GGA          GAT             CAA                GCC                   TCC                      ATC                         TCT                            TGC                               AGA                                  TCT                                     AGTCAG   AGC ATT    GTA       CAT          AGT             AAT                GGA                   AAC                      ACC                         TAT                            TTA                               GAA                                  TGG                                     TACCTG   CAG AAA    CCA       GGC          CAG             TCT                CCA                   AAG                      CTC                         CTG                            ATC                               TAC                                  AAA                                     GTTTCC   ATC CGA    TTT       TCT          GGG             GTC                CCA                   GAC                      AGG                         TTC                            AGT                               GGC                                  AGT                                     GGATCA   GGG ACA    GAT       TTC          ACA             CTC                AAT                   ATC                      AGC                         AGA                            GTG                               GAG                                  GCT                                     GAGGAT   CTG GGA    ATT       TAT          TAC             TGC                TTT                   CAA                      GGT                         TCA                            CAT                               GTT                                  CCG                                     TACACG   TTC GGA    GGG       GGG          ACC             AAG                CTG                   GAA                      ATA                         AAA                            C (Seq. ID No: 26)__________________________________________________________________________

                                  TABLE 23__________________________________________________________________________VH Nucleotide SequenceKC-4 VH (IIID-D9-JH3)__________________________________________________________________________ATG   GAC TTT    GGG       CTC          AGC             TTG                GTT                   TTC                      CTT                         GTC                            CTT                               ATT                                  TTA                                     AAAGGT   GTC CAG    TGT       GAA          GTG             CAG                ATG                   GTG                      GAG                         TCT                            GGG                               GGA                                  GTG                                     AAGCCT   GGA GGG    TCC       CTG          AAA             CTC                TCC                   TGT                      GCA                         GCC                            TCT                               GGA                                  TTC                                     GCTTTC   AGT AGC    TAT       GCC          ATG             TCT                TGG                   GTT                      CGC                         CAG                            GAG                               AAG                                  AGG                                     CTGGAG   TGG GTC    GCA       GAA          ATT             AGT                AGT                   GGT                      GGT                         AAT                            TAC                               GCC                                  TAC                                     TATCAA   GAC ACT    GTG       ACG          GGC             CGA                TTC                   ACC                      AGA                         GAC                            AAT                               GCC                                  AAG                                     AACACC   CTG TAC    CTG       GAA          ATG             AGC                AGT                   CTG                      AGG                         TCT                            GAG                               GAC                                  ACG                                     GCCATG   TAT TAC    TGT       GCA          AGG             GAG                GGT                   ATC                      CCG                         GCC                            TGG                               TTT                                  GCT                                     TACTGG   GGC CAA    GGG       ACT          CTG             GTC                TCT                   GTC                      TCT                         GCA                            G (Seq. ID No: 27)__________________________________________________________________________
Example 27

Amino Acid Sequences of KC-4 Chimeric Antibody Fv Regions

After the KC-4 Fv region cDNAS were cloned, and sequenced, and their cDNA-derived amino acid sequence was compared with the N-terminus sequence directly determined by a single run of amino acid sequencing on purified KC-4 antibody.

The cDNA sequences were shown to be accurate since in both cases they were identical for clones that were prepared from independent reverse transcription reactions. This confirms that the cloned cDNAs are authentic KC-4 Fv regions. The sequences are shown in Tables 24 and 25 below.

              TABLE 24______________________________________VL Amino Acid SequencesKC-4VL (kII-Jk2)______________________________________ MKLPVRLLVLMFWIPASSS (Seq. ID No: 28)FR1   DVLMTQTPLSLPVSLGDQASISC (Seq. ID No: 29)CDR1  RSSQSIVHSNGNTYLE (Seq. ID No: 30)FR2   WYLQKPGQSPKLLIY (Seq. ID No: 31)CDR2  KVSIRFS (Seq. ID No: 32)FR3   GVPDRFSGSGSGTDFTLNISRVEAEDLGIYYC (Seq. ID No: 33)CDR3  FQGSHVPYT (Seq. ID No: 34)FR4   FGGGTKLEIK (Seq. ID No: 35)______________________________________

              TABLE 25______________________________________VH Amino Acid SequencesKC-4VH (IIID-D9-JH3)______________________________________ MDFGLSLVFLVLILKGVQC (Seq. ID No: 36)FR1   EVQMVESGGGLVKPGGSLKLSCAASGFAFS (Seq. ID No: 37)CDR1  SYAMS (Seq. ID No: 38)FR2   WVRQSPEKRLEWVA (Seq. ID No: 39)CDR2  EISSGGNYAYYQDTVTG (Seq. ID No: 40)FR3   RFTISRDNAKNTLYLEMSSLRSEDTAMYYCAR (Seq. ID No: 41)CDR3  EGIPAWFAY (Seq. ID No: 42)FR4   WGQGTLVSVSA (Seq. ID No: 43)______________________________________

The sequences were interpreted as described by Kabat et al. (1991), supra. The residues that are underlined correspond to PCR primers. The mature VL and VH chains begin at amino-acids D and E of framework 1 (FR1), respectively.

Framework and CDR protein segments were identified according to Kabat et al. (1991), supra. VL is a group II κ chain.

Part of the CDR 3 and all of the framework 4 (FR4) are encoded by Jk2. VH belongs to group IIId. CDR 3 and FR4 resulted from a genomic recombination involving minigenes D9 and JH3. There is an asparagine glycosylation site in the light chain in FR3. The site reads NIS (Asn IIe Ser).

Example 28

Comparison of cDNA deduced Amino Acid Sequence with Directly Determined N-Terminal Fragment sequence

A comparison between the cDNA-derived polypeptide sequence and the amino acid sequence determined directly on the purified KC-4 monoclonal antibody was undertaken. The results are shown in Table 26 below.

              TABLE 26______________________________________Comparison of cDNA-deduced with DireclyDetermined N-Terminal Amino Acid Sequences______________________________________FIRST BAND TOPVH, cDNA-deduced       EVQMVESGGGLVKPGGSLKLS (Seq. ID       No: 44VH, Protein sequence       EVQMVESGGGLVKPGGXLKLS (Seq. ID       No: 45)SECOND BANDVL, cDNA-deduced       DVLMTQTPLSLPVSLGDQASI (Seq. ID       No: 46)VL, Protein sequence       DVLMTQTPLSLPVXXGDQASI (Seq. ID       No: 47)THIRD BANDVL, cDNA-deduced       DVLMTQTPLSLPVSLGDQASI (Seq. ID       No: 48)VL, Protein sequence       DVLMTQTPLSLPVSLGDQASI (Seq. ID       No: 49)______________________________________ X uncertain or alternative calls.

A sample of KC-4 (approximately 190 ug) was reduced with 5% mercaptoethanol (65° C. for 15 min.), separated on three lanes of a 10% SDS polyacrylamide gel, and electroblotted onto a ProBlott membrane (Applied Biosystems, Foster City, Calif.) in 90% 30 mM CAPS pH11, 10% methanol, for 1 hour at 25 V and at 4° C. The transferred protein species were stained with Commassie Blue. 3 bands were seen in each lane, of which 2 migrated as expected for a heavy and light chain. The third band migrated above the light chain. Amino acid sequencing was performed directly on the immobilized bands by the Biotechnology Instrumentation Facility, University of California, Riverside. The amino acid sequence given here is the sequencer's best guess.

Example 29

Construction of Mouse-Human Chimeric KC-4 Antibody Genes

The vector used were described in Example 1 above. Restriction ends were added to the cDNAs in a set of PCR reactions, using primers JO20, 21, 22, and 24.

The pAG4622 light chain vector and the pAH4604 heavy chain vector were described in Example 12 above.

The new VH and VL DNA fragments with appropriate restriction ends were integrated into pAH4604 and pAG4622 as described in Example 12 above. The vectors were then electroporated (together) also as described in Example 12.

Example 30

Tissue Binding Studies

The supernatants from stable transfectants were assayed for the presence of the mouse-human chimeric KC-4 antibody as described in Example 13. The chimeric antibody secreted in the supernatant bound both HMFG and BEM very strongly. In addition, the supernatants containing mouse-human chimeric KC-4 antibody were used to stain human breast carcinoma tissue sections by using the immunoperoxidase immunohistochemical staining technique. The intensity of the staining was comparable to that obtained with the original murine monoclonal antibody.

The KC-4 monoclonal antibody is known to bind the human milk fat globule and the breast epithelial mucin. This binding specificity of the KC-4 monoclonal antibody was maintained after the recombinant procedure. The KC-4 chimeric antibody bound very strongly to the human milk fat globule and the breast epithelial mucin as determined by radioassay (Ceriani, et al., Breast Cancer Res. Trent. 15:161 (1990)). In addition, the KC-4 chimeric antibody bound several human breast tumors in histopathological sections in a manner comparable to the KC-4 murine monoclonal antibody, as detected by immunostaining described in Example 15 above. This specificity of binding demonstrated the retained binding reactivity of the variable regions of KC-4 by the polypeptide of the invention when attached to the human Fc fragment.

Example 31

Hybridoma Cell Deposits

The hybridoma cell lines expressing the BrE-3 and KC-4 mouse-human chimeric and fully humanized BrE-3 antibodies were deposited on Nov. 13, 1992 under the Budapest Treaty with the ATCC, 12301 Parklawn Drive, Md. 20852, and have been assigned Accession Nos. HB 11199 (Chimeric BRE-3 A1C10), HB 11201 (Chimeric KC-4 1E8), and HB 11200 (Humanized BrE-3 A1C10). Hybridomas expressing fully humanized, and half humanized/half chimeric KC-4 antibodies were deposited with the ATCC, on Sep. 23, 1993, and have been assigned Accession Nos. ATCC HB 11455 (Humanized HuKC-4-4V2), HB 11454 (Light chain humanized/heavy chain chimeric HuKc-4V1), and HB 11456 (Heavy chain humanized/light chain chimeric HuKC-4V3). Hybridomas expressing half humanized/half chimeric BrE-3 antibodies were deposited with the ATCC on Nov. 11, 1993, and have been awarded Accession Nos. HB 11486 (Light chain humanized/heavy chain chimeric BRE-3V1) and HB 11487 (light chain humanized/heavy chain chimeric HuBRE-3V3).

Example 32

Materials and Assays for Epitope Mapping

The specific details of the preparation of materials, cell lines, and techniques employed were disclosed by Peterson, J. A., et al. (Peterson, J. A., et al., "Molecular Analysis of Epitope Heterogeneity of the Breast Mucin", Breast Epithelial Antigens, Ed. Ceriani, R. L., Plenum Press, NY (1991)), the relevant text of which is incorporated herein by reference.

Overlapping peptide hexamers were synthesized onto the ends of polyethylene pins using an Epitope Scanning Kit (Cambridge Research Biochemicals, Cambridge, UK), which is based on a method originally described by Geysen, H. L., et al. (Geysen, H. L., et al., "Use of Peptide Synthesis to Probe Vital Antigens for Epitopes to a Resolution of a Single Amino Acid", P.N.A.S. (USA) 81:3998-4002 (1984)). The polyethylene pins were arranged in a 8×12 configuration that fits into a 96 well microtiter dish. The pins are supplied with an alanine attached to the ends to which the amino acids are added consecutively using pentafluorophenyl active esters of fluorenylmethyloxycarbonyl (Fmoc)-L-amino acids. Each consecutive overlapping hexamer differs from the previous one by a single amino acid and enough were synthesized to span the entire sequence of the peptide to be tested so that every combination of hexamer was present. Each monoclonal antibody was tested for binding to the synthetic peptides using an ELISA method with horse radish peroxidase-conjugated goat anti-mouse IgG (Promega, Madison, Wis.) and color development with 2,2'azinobis (3-ethylbenzothiazoline-6-sulfonic acid (Sigma, St. Louis, Mo.).

The hexapeptides starting with A, P, D, and T bind well to the antibodies (Hexamers 1 to 3 and 20), whereas the hexamers starting between these positions did not. The hexamers prepared are shown in Table 27 below. From the hexamer that each monoclonal antibody binds the linear amino acid sequence essential for its binding to the antigen may be deduced. For example, BrE-3 required the sequence TRP within the hexamer. Other monoclonal antibodies required other amino acid sequences (e.g., Mc5, TRPAP; Mc1, DTR; BrE-1, DTRP). BrE-2 also required TRP but its different specificity for normal and tumor tissue indicates that its epitope on the native antigen is different from BrE-3.

                                  TABLE 27__________________________________________________________________________Epitope Mapping of Repeat Peptide Breast MucinHex- * *         *           * *               * *       *mer   G V T S A P D T R P A P G S T A P P       A                                        H                                        G                                        V T S A P        D                                                T        R                                                P__________________________________________________________________________ 1           P D T R P A 2             D T R P A P 3               T R P A P G 4                 R P A P G S 5                   P A P G S T 6                     A P G S T A 7                       P G S T A P 8                         G S T A P P 9                           S T A P P       A10                             T A P P       A                                        H11                               A P P       A                                        H                                        G12                                 P P       A                                        H                                        G                                        V13                                   P       A                                        H                                        G                                        V T14                                           A                                        H                                        G                                        V T S15                                           H                                        G                                        V T S A16                                           H                                        V T S A P17                                           V T S A P        D18                                             T S A P        D                                                T19                                               S A P        D                                                T        R20                                                 A P        D                                                T        R                                                P__________________________________________________________________________
Example 33

Epitope Mapping

Five different monoclonal antibodies (Mc1, Mc5, BrE1, BrE2 and BrE3), were prepared using the human milk fat globule (HMFG) for immunization. All identified epitopes on the highly glycosylated large molecular weight breast mucin. By immunohistochemistry they appeared to recognize different epitopes since each had different tissue and tumor specificities (Peterson, J. A., et al., "Biochemical and Histological Characterization of Antigens Preferentially Expressed on the Surface and Cytoplasm of Breast Carcinomas Cells Identified by Monoclonal Antibodies Against the Human Milk Fat Globule", Hybridoma 9:221-235 (1990)). Each monoclonal antibody bound to a different spectrum of normal tissues and their specificities for different carcinomas were different. BrE2 and BrE3, however, were quite similar. In addition, by screening breast γgtII cDNA expression libraries with some of these monoclonal antibodies, cDNA clones were isolated that produced fusion proteins that bound all of them, while other cDNA clones bound just some (Larroca, D., et al., "High Level Expression in E. Coli of an Alternate Reading Frame of pS2 mRNA that Encodes a Mimotope of Human Breast Epithelial Mucin Tandem Repeat" Hybridoma 11(2):191-201 (1992)).

This binding to the fusion proteins indicated that the epitopes for these 5 monoclonal antibodies included the polypeptide portion of this glycoprotein. To confirm this the binding of these monoclonal antibodies to two synthetic polypeptide 20-mers (PDTRPAPGSTAPPAHGVTSA and APPAHGVTSAPDTRPAPGST) that spanned the tandem repeat consensus sequence was tested (Gendler, S. J., et al., "Cloning of Partial cDNA Encoding Differentiation and Tumor-Associated Mucin Glycoproteins Expressed by Human Mammary Epithelium", P.N.A.S. (USA) 84:6060-6064 (1987); Siddiqui, J., et al., "Isolation and Sequencing of a cDNA Coding for the Human DF3 Breast Carcinoma-Associated Antigen", P.N.A.S. (USA) 85:2320-2323 (1988)).

One was started at the beginning of the published 20 amino acid repeat (Gendler, S. J., et al. (1987), supra) unit, and the other was started in the middle. All five monoclonal antibodies bound to both synthetic peptides, as did DF3, a monoclonal antibody against breast carcinoma cells produced by others (Hull, S. R., et al., "Oligosaccharide Differences in the DF3 Sialomucin Antigen from Normal Human Milk and the BT-20 Human Breast Carcinoma Cell Line", Cancer Comm. 1:261-267 (1989)). Three other monoclonal antibodies (Ceriani, R. L. , et al., "Characterization of Cell Surface Antigens of Human Mammary Epithelial Cells with Monoclonal Antibodies Prepared Against Human Milk Fat Globule", Somat. Cell Genet. 9:415-427 (1982); Peterson, J. A., et al., "Biochemical and Histological Characterization of Antigens Preferentially Expressed on the Surface and Cytoplasm of Breast Carcinoma Cells Identified by Monoclonal Antibodies Against the Human Milk Fat Globule", Hybridoma 9:221-235 (1990)) against other components of the HMFG that do not cross-react with the breast mucin, Mc13, against a 70 KDa glycoprotein, and Mc3 and Mc8, against a 46 KDa glycoprotein do not bind to these synthetic peptides (data not shown).

Example 34

Humanization Approach for Preparation of Humanized BrE-3 (BrE-3 HZ)

The present humanization approach is based on Padlan, E. A., "Choosing the Best Framework to Use in the Humanization of an Antibody by CDR-Grafting: Suggestions from 3-D Sstructural Data", Antibody Engineering 2nd. Annual Conf. San Diego, Calif. (Dec. 16-17, 1991).

The fine specificity may be preserved in a "humanized" antibody only if the CDR structures, their interaction with each other, and their interaction with the rest of the variable domains can be maintained. (Padlan, E. A.(1991), supra). This requires the preservation of residues of the FRs which contact the CDRs, those which are involved in the VL -VH contact, and those which are buried and could influence the overall domain structure and the structure of the combining site.

By examination of murine Fab structures, for which atomic coordinates are available, the FRs which are probably "important" in maintaining the structure of the combining site may be determined (Padlan, E. A., 8th International Congress of Immunol., Budapest, Hungary, Abstracts p. 19 (Aug. 2-28, 1992)).

The specificity of an antibody depends on the CDR structures and sometimes, on some of its neighboring residues as well. These CDR structures, in turn, depend on contacts with framework amino acids and on the interaction of the VL and VH domains. Thus, to ensure the retention of binding affinity, not only the CDR residues must be preserved, but also those FR residues that contact either the CDRs or their opposite domains, as well as all buried residues, which give shape to the variable domains. The buried amino acids are placed in exactly the same positions in human and in murine frameworks (Padlan, E. A., "A Possible Procedure for Reducing the Immunogenicity of Antibody Variable Domains While Preserving Their Ligand-Binding Properties", Molecular Immunology 28:489-498 (1991)).

This approach was applied to design humanized analogues of the variable regions of BrE-3. The humanization or design of the exemplary analogue peptide provided herein was undertaken as follows. The identification of the residues, which are most probably "important" in preserving the combining site structure, permits the selection of the best human FR sequences to use in the "humanization" of the murine BrE-3 chimeric antibody of known structure or analogues peptides of the invention. The results of the analysis can be used also to predict which FRs should probably be retained in those cases where no three-dimensional structural data are available.

The present procedure used was designed to reduce the immunogenicity of antibody BrE-3, its chimeric derivatives or fragments thereof while preserving its antigen-binding properties of an antibody are primarily determined by its CDRs, the CDRs of antibody BrE-3 were "grafted" onto a human framework. In addition, the FRs in antibody BrE-3 that are judged as probably important in maintaining the combining-site structure, were retained also in the humanized molecule.

Example 35

Choice of a Mouse Model of Known Structure

The classification of the VH and VL domains of an antibody such as BrE-3 was done according to Kabat, E. A., et al. (Kabat, E. A., et al., "Sequences of Proteins of Immunological Interest" NIH (1991). The BrE-3 kappa chain VL domain belongs to group II and the VH domain belongs to group IIIc. A murine antibody was then found, whose structure had been determined, and whose variable regions belong to the same classes. The anti-fluorescyl murine antibody 4-4-20 shown in Table 1 above (Herron, J. N., et al., "Three-Dimensional Structure of a Fluorescein-Fab Complex Crystallized in 2-Methyl-2,4-Pentanediol", Proteins, 5:271-280 (1989) fits these requirements since, like BrE-3, it has VL and VH domains belonging to groups II and IIIC. Thus, the three-dimensional structures of antibodies BrE-3 and 4-4-20 should be similar, and BrE-3 may be modeled after 4-4-20.

Example 36

Choice of the Target Human Framework

The choice of the target human framework was not based on the similarity of the amino acid sequence of the entire framework, but strictly on the similarity at the residues that were judged to be structurally important according to the 4-4-20 model. That is, only amino acids that could be involved in contacts with CDR with the opposite chain, or amino acids whose side-chains were predicted to be inwardly pointed. The positions of these amino acids are shown in Tables 8 and 9 and also in Tables 2, 3, 4, 5 and 6 above. These position are as follows.

For the light chain variable region framework: 1, 2, 3, 4, 5, 6, 11, 13, 19, 21, 23, 35, 36, 37, 38, 44, 45, 46, 47, 48, 49, 58, 60, 61, 62, 69, 71, 73, 75, 78, 82, 86, 88, 98, 102, and 104.

For the heavy chain variable region framework: 4, 6, 12, 18, 20, 22, 24, 27, 28, 29, 30, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 66, 67, 68, 69, 71, 73, 76, 78, 80, 82c, 86, 88, 90, 91, 92, 93, and 94.

The numbering system is conventionally accepted (Kabat, et al. (1991), supra) and shown in Tables 10 and 11 above. In this case, the consensus sequences of all human Fv regions were selected as the target human framework to minimize the immunogenicity of the product.

First, the sequences of the mouse variable chains were aligned with consensus sequences from all known human variable region classes (Herron, J. N., (1989), supra) and the number of differences in the amino-acids that must be retained from the mouse were scored. The positions of these amino acids were obtained from those of murine monoclonal antibody 4-4-20, which was chosen to model BrE-3 as shown in Tables 28 and 29 below.

                                  TABLE 28__________________________________________________________________________Choice of BrE-3 VL target human framework__________________________________________________________________________BrE3 VL BRE3K ##STR6##HuKi-n.IQ...S.S..SA.V..RVT.T. .A..S..XXS-ISN..A .YQ..P.KA...... A..SLE.HuKii-nt.I....S......TP.EP..... ....S.L.SXD.....N .Y...P....Q.... LV.N.A.HuKiiiEI.L..S.GT.SL.P.ER.TL.. .A..SVSSS.----..A .YQ..P..A.R.... G..S.ATHuKiv.I....S.D..A....ER.T.N. K...SVLYSS.NKN..A .YQ..P..P...... W..T.E. BRE3K ##STR7##HuKi-n...S.......G.....T..SLQP..FAT.Y. Q.YNSL.EW. ..Q...V...HuKii-nt...........G..............V...Y. M.ALQX.RX. ..Q...V...HuKiii.I.........G.....T...L.P..FA..Y. Q.YGSS.PL. ..Q...V...HuKiv...........G.....T..SLQ...VA..Y. Q.YYST.-X. ..Q...V...__________________________________________________________________________ . identity with the murine sequence * the mouse residues that are structurally important.

                                  TABLE 29__________________________________________________________________________Choice of BrE-3 VH Target Human Framework__________________________________________________________________________Bre3 VH BRE3VH  ##STR8##HuHI  Q.Q.VQ..AEVKK..A.V.V..K... Y..T SYAIS-- ....A.GQ....MGHuHII Q.Q.Q...P...K.SQTLS.T.TV.. GSV. SYXWSWN .I..P.G.....IGHuHIII ..Q.V............LR....... .... SYA.S-- ....A.G......S BRE3VH EIRNCDR2KANNHATYYDESVKG *R*F*T*IS*RD*DSK*SR*VY*LQ*MIS*LRAE*DTGL*Y*Y* *HuHI  W.N-PYG.GD.N.AQRFQ. .V..TA.T.T.TA.MELS...S...AV...ARHuHII R.YYR.YSGS.K.NP.L.S .V...V.T..NQFS.KLS.VT.A..AV...ARHuHIII V.SG.TDGGS...AD.... .......N..NTL....N.......AV...AR BRE3VH  EF-------------CDR3--AN *WGQG*TL*VT*VSAHuHI  APGYGSGGGCYRGDYKFDY ..........SHuHII ELPGGYKGDDYYYKKGFDV ..........SHuHIII GRXGXSLSGXYYYYHYFDY ..........S__________________________________________________________________________ . identity with the murine sequence * the mouse residues that are structurally important.

Based on these scores, the human frameworks belonging to groups Vk II and VH III were chosen to receive the BrE-3 CDRs plus other important amino acids.

Example 37

Identification of Murine/Human Differences

The original mouse sequences (BrE-3 VK or VH) were aligned with their closest human (Human KII or HIII) relatives that were chosen after comparing their sequences in Table 28 above. The alignment of these two sequences is shown in Table 30 below. The information in this table is also contained in Table 28 above, but is reproduced here for clarity. The CDRs are not shown, since their sequences were not changed during the humanization process.

Thus, Table 30 shows the maximum number of amino acids that can be changed toward the humanization of BrE-3, based on the consensus human sequences obtained from the current databases (Kabat, E. A., et al. (1991), supra). If all these positions were to be replaced with the corresponding human amino acids, the corresponding CDR grafted antibody variable regions would be attained.

                                  TABLE 30__________________________________________________________________________Corresponding Amino Acid Sequences of VK BrE-3 and HUman__________________________________________________________________________Kll BrE-3 VKCDR2--------------- WFLQKSGQSPKLLIYHuman KII--------------- .Y...P....Q.... BrE-3 VK-CDR3-------- FGGGTKLEIKISRVEAEDLGVYFCHuman KII--------- ..Q...V...............V...Y. BrE-3 VHCDR1---- WVRQSPEKGLEWVAMKLSCAASGFTFSHuman III---- ....A.G......S....LR...........BrE-3 VHCDR2-------------- RFTISRDDSKSRVYLQMISLRAEDTGLYYCTGHuman III .......N..NTL....N.......AV...AR BrE-3 VH CDR3------------------- WGQGTLVTVSAHuman III ..........S__________________________________________________________________________

Table 31 and 32 below contain the same information as Table 30 above in a different format. It shows the numbers of the residues that would have to be changed in order to completely convert the original murine framework completely into a human consensus framework.

              TABLE 31______________________________________BrE-3 VL Amino Acid Candidates for Changeinto Human Consensus SequencesVL______________________________________VI2             FY36        EG68GQ100TS7             SP40        LV83LV104ST14            KQ45        FY87LP15DE17QP18______________________________________

              TABLE 32______________________________________BrE-3 VH Amino Acid Candidates for Changeinto Human Consensus SequencesVH______________________________________KQ3             SA40        DN73AS113EV5             EG42        SN76ML18            AS49        RT77KR19                        VL78                       IN82a                       GA88                       LV89                       TA93                       GR94______________________________________
Example 38

Identification of important murine amino-acids

The "important" murine amino acids that should be preserved were chosen based on the contacts of a particular amino acid with the CDRs, and with the opposite chains and/or whether their side chains are pointing inwardly or outwardly. The positions of these "important" amino acids were determined based on the examination of the known structures of other antibodies. This information is provided in Tables 33 and 34 below.

              TABLE 33______________________________________Important VL Amino Acid Positions to be PreservedVL______________________________________V2        CDR contact      BuriedF36       CDR contact      Contact with VHK45       CDR contactF87       Possible contact with VHG100      Possible contact with VHL104      Buried______________________________________

              TABLE 34______________________________________Important VH Amino Acid Positions to be PreservedVH______________________________________M18       BuriedS40       BuriedA49       CDR contact          BuriedD73       CDR contactS76                            BuriedV78       CDR contact          BuriedL89       Might affect interaction with VLT93       CDR contactG94       CDR contact          Buried______________________________________

Most of the "important" amino acids were selected on the basis of the structure of antibody 4-4-20 and according to Tables 2, 3, 4, 5, 6, 8 and 9 above. Two important amino acids out of each chain, however, were selected based on more general structural analysis, using other antibody structures. This was done to maximize the chances of conserving ligand binding properties. In particular the preservation of the Leu at 89-H was suggested in order to ensure the maintenance of, the VL :VH contact. Although the residue at 89-H is usually not in contact with VL and is only partly buried, it nonetheless contributes to the interface. A Val for Leu replacement at this position could very well create a "cavity" which could affect the contact. An IIe for Leu replacement would probably be fine since these amino acids have essentially the same side chain volume.

Finally, by comparing the position of all amino acids that are candidates for mutation, shown in Tables 31 and 32 above, with those that are "important" and should be preserved, shown in Tables 33 and 34, the final selection of amino acid positions for actual mutation was attained. Any "important" amino acid position was eliminated from the list of candidates. Table 35 below shows the amino acids that were selected for changed from murine to human identities to obtain the present humanized analogue.

              TABLE 35______________________________________Selected Amino Acids for Mutation   VL      VH______________________________________   TS7          KQ3   ST14         EV5   LP15         KR19   DE17         EG42   QP18   RT77   SP40         IN82a   EG68         GA88   LV83         AS113______________________________________
Example 39

Introduction of Changes in Amino Acid Sequence

The changes were done at the DNA level in sequential manner. All but one of the codon mutations were performed using enzymatic inverse PCR (EIPCR), a mutagenesis technique developed by Stemmer and Morris (Stemmer, W. P. C and Morris, S. K., "Ezymatic Inverse Polymerase Chain Reaction: a Restriction Site Independent, Single Fragment Method for High Efficiency Site-Directed Mutagenesis", BioTechniques 13:146-220 (1992)).

First, the entire plasmid, containing the target cDNA was amplified by inverse PCR using terminal mutagenic oligonucleotides.

Second, Bsal was used to cut the ends of the incorporated primers. This enzyme cuts at a site that is displaced from its recognition site. Thus, after digestion of the open amplified plasmid with Bsal, the Bsal recognition sequence was removed from the ends of the DNA. The DNA was left with complementary sticky ends and can be closed into a functional plasmid that contains the mutagenized region. The amino acid and DNA sequences of the non-mutated (wild-type) variable light and heavy chains of BrE-3 are shown in Tables 15 and 16 above. The amino acid sequences of the BrE-3 frameworks and the mutations that were performed for the humanization, the oligonucleotide that was used for the mutagenesis, and the method of mutagenesis are shown in Tables 36 to 44 below.

                                  TABLE 36__________________________________________________________________________FVL FR1 Mutation Sites        Analogue 1              DNA Codon                      Method forPosition    FR1      (8 Changes)              (FR1) (Analogue)                      Mutagenesis                            Primers__________________________________________________________________________Leader    Peptide Not Shown    D    V    V    M    T    Q7   T        S     ACT TCT EIPCR*                            (JO37, JO38)    P    L    S    L    P    V14  S        T     AGT ACT EIPCR (JO37, JO38)15  L        P     CTT CCT EIPCR (JO37, JO38)    G17  D        E     GAT GAG EIPCR (JO37, JO38)18  Q        P     CAA CCA EIPCR (JO37, JO38)    A    S    I    S    C__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 37__________________________________________________________________________FVL FR2 Mutation Sites     Analogue 1           DNA Codon                   Method forPosition    FR2   (8 Changes)           (FR2) (Analogue)                   Mutagenesis                         Primers__________________________________________________________________________CDR1    Not Shown    W    F    L    Q    K40  S     P     TCA CCA EIPCR*                         (JO39, JO40)    G    Q    S    P    K    L    L    I    Y__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 38__________________________________________________________________________FVL FR3 Mutation Sites     Analogue 1           DNA Codon                   Method forPosition    FR3   (8 Changes)           (FR3) (Analogue)                   Mutagenesis                         Primers__________________________________________________________________________CDR2    Not Shown    G    V    P    D    R    F    S    G    S    G    S68  E     G     GAG GGG EIPCR*                         (JO41, JO42)    T    D    F    T    L    K    I    S    R    V    E    A    E    D83  L     V     CTG GTG EIPCR (JO41, JO42)    G    V    Y    F    C__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 39__________________________________________________________________________FVL FR4 Mutation Sites     Analogue 1           Aanlogue 2                 DNA Codon                         Method forPosition    FR4   (10 Changes)           (6 Changes)                 (FR4) (Analogue)                         Mutagenesis                               Primers__________________________________________________________________________CDR3    Not Shown    F     F    G     G    G     G    G     G    T     T    K     K    L     L    E     E    I     I    K     K__________________________________________________________________________

                                  TABLE 40__________________________________________________________________________FVH FR1 Mutation Sites        Analogue 1              DNA Codon                      Method forPosition    FR1      (8 Changes)              (FR1) (Analogue)                      Mutagenesis                            Primers__________________________________________________________________________Leader    Peptide Not Shown    E    V3   K        Q     AAG CAG EIPCR*                            (JO57, JO58)    L5   E        V     GAG GTG EIPCR (JO57, JO58)    E    S    G    G    G    L    V    Q    P    G    G    S    M19  K        R     AAA AGA EIPCR (JO57, JO58)    L    S    C    A    A    S    G    F    T    F    S__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 41__________________________________________________________________________FVH FR2 Mutation Sites     Analogue 1           DNA Codon                   Method forPosition    FR2   (8 Changes)           (FR2) (Analogue)                   Mutagenesis                         Primers__________________________________________________________________________CDR1    Not Shown    W    V    R    Q    S    P42  E     G     GAG GGG EIPCR*                         (JO55, JO56)    K    G    L    E    W    V    A__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 42__________________________________________________________________________FVH FR3 Mutation Sites     Analogue 1           DNA Codon                   Method forPosition    FR2   (8 Changes)           (FR2) (Analogue)                   Mutagenesis                         Primers__________________________________________________________________________CDR2    Not Shown    R    F    T    I    S    R    D    D    S    K    S77  R     T     AGA ACT EIPCR*                         (JO53, JO54)    V    Y    L    Q    M82  I     N     ATA AAT EIPCR (JO53, JO54)    S    L    R    A    E    D    T88  G     A     GGC GCC EIPCR (JO53, JO54)    L    Y    Y    C    T    G__________________________________________________________________________ *EIPCR: Enzymatic Inverse Polymerase Chain Reaction

                                  TABLE 43__________________________________________________________________________FVH FR4 Mutation Sites     Analogue 1           DNA Codon                   Method forPosition    FR2   (8 Changes)           (FR2) (Analogue)                   Mutagenesis                         Primers__________________________________________________________________________CDR3    Not Shown    W    G    Q    G    T    L    V    T    V    S113 A     S     GCA TCT PCR+                         (JO51, JO52)*__________________________________________________________________________ + * PCR: Polymerase Chain Reaction

                                  TABLE 44__________________________________________________________________________Sequences of Mutagenic Oligonucleotides__________________________________________________________________________JO37 TCC CTG GGT CTC ACT CCT GGA GAG CCA GCT TCC ATC TCT TGC AGA TCT AGT(Seq. ID No. 50)JO38 AGC TTG GGT CTC AGG AGT GAC AGG CAG GGA GAG TGG AGA TTG GGT CAT CACAAC (Seq. ID No. 51)JO39 G TTC CTG GTC TCG CCA GGC CAG TCT CCA AAG CTC CTG (Seq. ID No. 52)JO40 T TGG AGG TCT CCC TGG CTT CTG CAG GAA CCA ATA TAA AT (Seq. ID No.53)JO41 TTC ACA GGT CTC ATC AGC AGA GTG GAG GCT GAG GAT GTG GGA GTT TAT TT(Seq. ID No. 54)JO42 AGC CTC GGT CTC GCT GAT CTT GAG TGT GAA ATC TGT CCC TGA TCC ACT GC(Seq. ID No. 55)JO51 CCT GGA GGA TCC ATG AGA CTC TCT TGT GCT (Seq. ID No. 56)JO52 GTT GGG GCT AGC AGA AGA GAC AGT GAC CAG AGT (Seq. ID No. 57)JO53 TAC CTG GGT CTC AAT AGC TTA AGA GCT GAA GAC ACT GCC CTT TAT TAC TGT(Seq. ID No. 58)JO54 TTC AGC GGT CTC GCT ATT CAT TTG CAG GTA CAC AGT ACT TTT GGA ATC ATC(Seq. ID No. 59)JO55 GTC CGC GGT CTC CCA GGG AAG GGG CTT GAG TGG GTT GCT GAA ATT AGA AA(Seq. ID No. 60)JO56 CTC AAG GGT CTC CCC TGG AGA CTG GCG GAC CCA GTC CAT CCA GGC ATC A(Seq. ID No. 61)JO57 T GAG GAG GTC TCA GGA GGC TTG GTG CAA CCT GGA GGA TCC ATG AGA CTCTCT (Seq. ID No. 62)JO58 GG TTG CGG TCT CCC TCC TCC AGA CTC CAC AAG CTG CAC TTC ACT CTG GAC A(Seq. ID No. 63)__________________________________________________________________________ *note Primer set JO51 and JO52 was originally intended for mutagenizing K19 to R (by JO51) and A113 to S (by JO52), as described below. Primer JO51, however, was somehow defective (it did not run as a single band on polyacrylamide gel) and thus only the A113 to S mutagenesis was successful. Mutation K19 to R was accomplished at a latter time with EIPCR, using primers JO57 and JO58.
Example 40

Synthesis of Primers

All primers except JO51 and JO52 were synthesized on a PCR-Mate EP DNA synthesizer model 391 (Applied Biosystems, Foster City Calif.). Primers JO51 and JO52 were purchased from Keystone Laboratories, Inc, Menlo Park, Calif. Both, the PCR method used with JO51 and JO52 and the EIPCR method used with all other primer sets, are described below. The EIPCR method was used for preparing all primer sets with the exception of JO51 and JO52. This region is part of a larger cDNA fragment that encodes the variable region of the antibody and that is inserted into the 3 Kb plasmid pCR1000 (Invitrogen Corporation, San Diego, Calif.).

The plasmid DNA template was extracted with a kit purchased from QIAGEN (Tchapsworth, Calif.) and diluted to 1 ng/μl in 10 mM TRIS 1 mM EDTA pH 7.5-8. This plasmid is composed of vector pCR1000 (Invitrogen Corporation, San Diego, Calif.) into which the cDNA encoding the variable region to be humanized was inserted.

A mixture of PCR primers was made where each primer was present at a concentration of 10 pmole/μl in water.

The PCR amplification conditions were as follows. All reagents as well as the GeneAmp PCR system 9600 were purchased from Perkin Elmer Cetus. Optimal PCR conditions were determined empirically for each pair of mutagenic primers. A matrix of conditions varying the concentration of MgC12, mutagenic primers, and template plasmid DNA were set up. The annealing and extension temperatures during PCR may be varied.

Plasmid template (500 pg/μl), 0.5 μM each mutagenic oligonuceotide, 1 mM MgCl2, 10 mM TRIS pH 8.3, 50 mM KCl, 0.2 mM each nucleotide triphosphate (dGTP, dATP, TTP, dCTP), and Taq polymerase 1 unit/ 20 μl.

Example 41

Hot Start PCR

All the components of the PCR mixture, with the exception of Taq polymerase, were mixed in a 95 μl volume. The mixture was then dispensed in 19 μl aliquots into 5 PCR tubes. The reason for performing five independent reactions was to decrease the odds that unwanted mutations be isolated as a result of nucleotide misincorporation during PCR. The tubes were heated to 95° C. for 5 Minutes and then cooled to 72° C. While at that temperature 1 μl of an appropriate Taq polymerase dilution in 10 mM TRIS pH 8.3, 50 mM KCl was added to the reaction tubes. The temperature cycling then proceeds as follows.

94° C., 3 min

(94° C., 1 min) (50° C., 1 min) (72° C., 3.5 min)! 3 cycles

(94° C., 1 min) (55° C., 1 min) (72° C., 3.5 min)! 25 cycles

72° C., 10 min

Example 42

Extra Final Extension

After cycling the contents of the five tubes were mixed an extra final extension reaction was carried out. Extra nucleotide triphosphates (to 125 μM) were added, 5 units of Taq polymerase were also added and the mixture was heated to 72° C. for 10 minutes.

Example 43

Purification of PCR Products

The PCR products were then separated on a 0.8% agarose gel in 1XTAE buffer and 0.5 μg/ml Ethidium Bromide. The correct DNA band was visualized with UV light (366 nm), excised from the gel and extracted with the GeneClean kit purchased from Bio 101, La Jolla, Calif.

Example 44

Restriction Digestion

The DNA was then digested with Bsal for two hours at 60 degrees celsius in 25 μl (20.5 μl of DNA, 2.5 μl 10×buffer 4 (NEB) and 2 μl BsAI (NEB). Bsal sites were designed near the 5' end of the PCR primers. The primers included 6 extranucleotides 5' of the Bsal sites to facilitate digestion by Bsal. There were no Bsal sites elsewhere in the plasmid. If there were other restriction enzymes may be used as adviced by Stemmer and Morris (Stemmer and Morris (1992), supra)). This special class of restriction enzyme cuts at a site that is different from its recognition site but, nevertheless, at a precise distance from it. Using this method, there was no need for having restriction sites in the sequence in order to perform the mutagenesis.

Example 45

Second Purification

The restricted products were then separated on a 0.8 % agarose gel in 1×TAE buffer and 0.5 μg/ml Ethidium Bromide. The correct DNA band was visualized with UV light (366 nm), excised from the gel and extracted with the GeneClean kit purchased from Bio 101, La Jolla, Calif.

Example 46

Ligation (Reclosure of Plasmid)

The ligation mixtures consisted of 5 μl extracted DNA, 2 μl 10×ligation buffer (NEB) 1 μl T4 DNA polymerase (NEB), 12 μl water. The amount of plasmid DNA may be varied depending of the intensity of the band extracted from the Gel. Ligation was carried out at room temperature for 2 hours, or alternatively at 14° C. overnight.

Example 47

Transformation and Sequencing

The reclosed plasmids were then transformed into E. coli. We used Inv alpha F' competent cells purchased from Invitrogen Corporation, San Diego Calif.

Plasmid DNA was then prepared from a few transformants and sequenced to verify that mutagenesis was succesful.

Example 48

Mutagenesis using JO51 and JO52

These oligonucleotides were designed to mutagenize K19 to R (by JO51) and A113 to S (by JO52), not by using EIPCR but using the normal PCR (with primers pointing to each other). Primer JO51 carried a BamHI site and primer JO52 carried an NheI site. After mutagenenic PCR amplification the resulting amplified DNA cassette was inserted into the plasmid in lieu of the corresponding wild-type DNA fragment. There was not compeling reason to use this method over the EIPCR method except that conveniently placed restriction sites (BamHI and NheI) were available. This method, however, yielded only the A113 to S mutation. A subsequent analysis showed that the JO51 primer, which carried the K19 to R mutation ran aberrantly on a polyacrylamide gel.

The protocol for the mutagenic amplification step was as follows. Plasmid template (500 pg/μl), 0.75 μM each mutagenic oligonuceotide, 2 mM MgCl2, 10 mM TRIS pH 8.3, 50 mM KCl, 0.2 mM each nucleotide triphosphate (dGTP, dATP, TTP, dCTP), and Taq polymerase 1 unit/ 20μl.

The PCR was hot started as described for EIPCR above. The temperature cycling conditions used were as follows.

94° C., 3 min

(94° C., 1 min) (44° C., 1 min) (72° C., 1 min)! 3 cycles

(94° C., 5 sec) (65° C., 1 min) (72° C., 1 min)! 32 cycles

72° C., 10 min

The extra final extension and purification of PCR products were conducted as described for EIPCR above.

The restriction digestion of the vector and the insert were conducted as follows. The PCR product was digested with BamHI and NheI for 1 h 50' at 37° C. (19 μl of DNA, 2.5 μl 10×buffer 3 (NEB), 1.5 μl BamHI (NEB), 1.5 μl NheI (NEB). The vector, which is the starting plasmid described above, was digested under similar conditions 1μl plasmid (1 μg), 2 μl 10×buffer 3 (NEB), 1.5 μl BamHI (NEB), 1.5 μl NheI (NEB) 14 μl water, 2 h 30' at 37° C!.

The restricted products, vector and insert, were then purified once more as described above for EIPCR.

The ligation of the fragments was conducted as follows. The ligation mixtures consisted of 5 μl vector, 5 μl insert, 2 μl 10×ligation buffer (NEB) 1 μl T4 DNA polymerase (NEB), 7 μp water. The amount of plasmid DNA may be varied depending of the intensity of the band extracted from the Gel. Ligation is carried out at 14° C. overnight. A control ligation with vector only was carried out in parallel.

The transformation of the host cells was conducted as described for EIPCR.

Example 49

Plasmid Preparation and Sequencing

Plasmid DNA was then prepared from several independent transformants. A few of the plasmids that were shown by restriction analysis to contain the insert were then sequenced to verify that the mutagenesis was successful. When one of the sequenced DNAs contained the desired mutation it was utilized for the next mutation cycle. The fully mutated humanized analogue BrE-3 DNA sequences for the VH and VL segments are shown in Tables 45 and 46 below.

                                  TABLE 45__________________________________________________________________________BrE-3 VL Humanized Analogues DNA SequencesBrE-3 VL FR-HZ__________________________________________________________________________ATG   AAG TTG    CCT       GTT          AGG             CTG                TTG                   GTG                      CTG                         TTG                            TTC                               TGGATT   CCT GCT    TCC       ATC          AGT             GAT                GTT                   GTG                      ATG                         ACC                            CAA                               TCTCCA   CTC TCC    CTG       CCT          GTC             ACT                CCT                   GGA                      GAG                         CCA                            GCT                               TCCATC   TCT TGC    AGA       TCT          AGT             CAG                AAC                   CTT                      GTA                         CAC                            AAC                               AATGGA   AAC ACC    TAT       TTA          TAT             TGG                TTC                   CTG                      CAG                         AAG                            CCA                               GGCCAG   TCT CCA    AAG       CTC          CTG             ATT                TAT                   AGG                      GCT                         TCC                            ATC                               CGATTT   TCT GGG    GTC       CCA          GAC             AGG                TTC                   AGT                      GGC                         AGT                            GGA                               TCAGGG   ACA GAT    TTC       ACA          CTC             AAG                ATC                   AGC                      AGA                         GTG                            GAG                               GCTGAG   GAT GTG    GGA       GTT          TAT             TTC                TGC                   TTT                      CAA                         GGT                            ACA                               CATGTT   CCG TGG    ACG       TTC          GGT             GGA                GGC                   ACC                      AAG                         CTG                            GAA                               ATCAAA   C (Seq. ID No. 64)__________________________________________________________________________

                                  TABLE 46__________________________________________________________________________BrE-3 VH Humanized Analogue DNA SequenceBrE-3 VH FR-HZ__________________________________________________________________________ATG   TAC TTG    GGA       CTG          AAC             TAT                GTC                   TTC                      ATA                         GTT                            TTT                               CTCTTA   AAA GGT    GTC       CAG          AGT             GAA                GTG                   CAG                      CTT                         GTG                            GAG                               TCTGGA   GGA GGC    TTG       GTG          CAA             CCT                GGA                   GGA                      TCC                         ATG                            AGA                               CTCTCT   TGT GCT    GCT       TCT          GGA             TTC                ACT                   TTT                      AGT                         GAT                            GCC                               TGGATG   GAC TGG    GTC       CGC          CAG             TCT                CCA                   GGG                      AAG                         GGG                            CTT                               GAGTGG   GTT GCT    GAA       ATT          AGA             AAC                AAA                   GCC                      AAT                         AAT                            CAT                               GCAACA   TAT TAT    GAT       GAG          TCT             GTG                AAA                   GGG                      AGG                         TTC                            ACC                               ATCTCA   AGA GAT    GAT       TCC          AAA             AGT                ACT                   GTG                      TAC                         CTG                            CAA                               ATGAAT   AGC TTA    AGA       GCT          GAA             GAC                ACT                   GCC                      CTT                         TAT                            TAC                               TGTACT   GGG GAG    TTT       GCT          AAC             TGG                GGC                   CAG                      GGG                         ACT                            CTG                               GTCACT   GTC TCT    TCT G (Seq. ID No.: 65)__________________________________________________________________________
Example 50

Antibody Expression

Two expression vectors pAG4622 and pAH4604 (Coloma, M. J., et al. (1992), supra) were used that were developed and provided by S. L. Morrison (Dept. of Microbiology and Molecular Genetics, UCLA). Any cDNA encoding a signal peptide and either the variable heavy chain or the variable light chain can, in principle, be inserted into these vectors resulting in a construction that encodes an IgG1, K, antibody with human constant regions. Correctly modified cDNAs were excised from pCR1000 with EcoRV and Sal I and inserted into pAG4622. These encode the modified light chain. The wild-type heavy chain was similarly excised from pCR1000 by digestion with EcoRV and NhEI and inserted into pAH4604. The restriction and ligation reactions necessary to accomplish these operations were performed under the conditions stipulated by the enzyme manufacturers (New England Biolabs, Beverly Mass.). Both the vectors and the inserts were purified from an agarose gel prior to ligation, using the Bio101 (La Jolla, Calif.) GeneClean kit (glass beads). The VH and VL regions in the final constructions were sequenced once again to verify that they are correct. The non-producer myeloma cell line SP2/0-Ag14, ATCC: CRL 1581, (Shulman M., et al. (1978), supra) was transfected with both plasmid constructions, and antibody producers are isolated following the recommendations outlined in (Coloma, M. J. et al. (1992), supra) except that selection was done only for the uptake of hisD (by adding 5 mM histidinol to the medium and readjusting the pH to 7.4 with NaOH). Usually after ten days, stable transfectant colonies were established at a frequency of approximately 10-5 to 10-4. Colonies were then transferred to normal medium (without histidinol). The culture media were either Dulbeco's modified Eagle's medium (DME): fetal bovine serum (FBS), 90:10, v/v, or a mixture of DME:RPMI:FBS, 45:45:10, v/v/v. Penicillin and streptomycin were added to prevent bacterial growth.

The supernatants from stable transfectants were assayed for the presence of the antibodies. This was done by capturing the secreted chimeric antibody with a plate-bound goat anti-human-kappa chain antibody and developing with goat anti-human-gamma chain antibody, essentially as described previously (Coloma, M. J. (1992), supra) except that the secondary antibody was radiolabeled with 125 I. The supernatants were also assayed for binding to human milk fat globule (HMFG) as described previously (Ceriani R. L., et al., "Diagnostic Ability of Different Human Milk Fat Globule Antigens in Breast Cancer", Breast Cancer Res. Treat., 15:161-174 (1990)). HMFG is bound to the microtiter plates as described previously (Ceriani, R. I. (1984), supra). Usually most colony supernatants were positive by both assays.

Colonies that secrete the highest level of antibody in the supernatants, as determined by these assays, were subcloned and subsequently adapted to serum-free medium for the purification of antibody. Serum free medium contains HL-1 supplement as directed by the manufacturer (Ventrex Labs., Portland, Me.).

Example 51

Half Humanized/Half Chimeric BrE-3 Antibody

A humanized BrE-3 light chain was paired with a non-humanized BrE-3 chimeric heavy chain by co-transfection of SP2/0 myeloma cells with hybrid plasmids carrying the respective DNA sequences and those of a human Fc (HuBrE-3V1; HuBrE-3V3).

Example 52

Determination of Affinity Constants

The secreted half humanized/half chimeric and fully humanized antibodies were purified from culture supernatants using a Sepharose 4B-protein A column (Bio-Rad, Richmond, Calif.) as described by Ey, P. L., et al. (Ey, P. L., et al. (1978), supra). Microtiter plates (Dynatech, Chantilly, Va.) were prepared as described by Ceriani, R. L., et al. (Ceriani, R. L., et al. (1992), supra) using successive layers of methylated BSA, glutaraldehyde, anti-,β-galactosidase and the bacterial fusion protein 11-2 (a hybrid of β-galactosidase and human mammary mucin). Each well contained 388 ng of the 11-2 fusion protein. To each well were added 25 μl 125 I -BrE-3 in RIA buffer (10 % bovine calf serum, 0.3% triton X-100, 0.05 % sodium azide pH7.4, in phosphate buffer saline) and compete with 25 μl of either unlabeled murine or chimeric antibody in RIA buffer at the final concentrations of 130 pM, 850 pM, 1.3 nM, 4 nM, and 13 nM). Iodinations were performed with 125 I (17 Ci/mg, Nordion International). Fifty micrograms of monoclonal antibody BrE-3 (Coulter, Hialeah, Fla.) were labeled at a specific activity of 9.56 mCi/mg using the chloramine T method as described previously by Ceriani, R. L., et al. (Ceriani, R. L., et al. (1988), supra).

Antibody-antigen affinity constants were determined by taking the reciprocal of the concentration of competing unlabeled monoclonal antibody that produced 50% binding as described by Sheldon, K., et al. (Sheldon, K., et al. (1987), supra). The protocol used to determine affinity constants was as described above except that in each case, an unlabeled antibody competed for binding to antigen against the same radiolabeled antibody. Both, the half humanized/half chimeric antibody and the fully humanized antibody competed about as well or better with the murine BrE-3 antibody for the antigen.

Polyacrylamide gel electrophoresis was performed to insure that the antibody chains migrated as expected.

The affinity binding constants of the murine, chimeric, half humanized and humanized antibodies were determined in independent competition assays.

Example 53

Histochemical Specificity

Immunohistochemical staining using the immunoperoxidase technique of consecutive human breast carcinoma tissue sections was used as a test to verify that the analogue antibodies retain useful affinity for the carcinoma antigens. Breast carcinoma tissue sections were stained with the supernatant of the half humanized/half chimeric and fully humanized transfected cells using the Vectastain ABC method (Vector Labs, Burlingame, Calif.). Both antibodies showed strong staining patterns.

Example 54

Gel Chromatography of Half Humanized/Half Chimeric and Fully Humanized Antibodies

Antibody disulfide bonds were reduced by heating for 10 min at 65° C. in Laemmli loading buffer containing 5% beta-mercaptoethanol. The separated chains were then chromatographed on a SDS polyacrylamide gel (10%). Two bands were observed for both half humanized/half chimeric antibodies of similar migration pattern as the murine antibody. These data were also confirmed by Western blotting.

Example 55

Deduced Amino Acid Sequences of VL -HZ and VH -HZ

The amino acid sequences of the light and heavy chains of the analogue humanized antibody are shown in Tables 47and 48 below. These amino acid sequences may be improved either to increase affinity for the antigen or to decrease immunogenicity in humans. Numerous variants of this sequence may be engineered in accordance with the invention.

              TABLE 47______________________________________BrE-3 VL Analogue Amino Acid SequenceBrE-3VL FR-HZ______________________________________Leader mklpvrllvlLFWIPASIS (Seq. ID No.: 66)FR1    DVVMTQSPLSLPVTPGEPASISC (Seq. ID No.: 67)CDR1   RSSQNLVHNNGNTYLY (Seq. ID No.: 68)FR2    WFLQKPGQSPKLLIY (Seq. ID No.: 69)CDR2   RASIRFS (Seq. ID No.: 70)FR3    GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC  (Seq. ID No.: 71)CDR3   FQGTHVPWT (Seq. ID No.: 72)FR4    FGGGTKLEIK (Seq. ID No.: 73)______________________________________

              TABLE 48______________________________________BrE-3 VH Analogue Amino Acid SequenceBrE-3VH FR-HZ______________________________________Leader mylglnyvflVFLLKGVQS (Seq. ID No.: 74)FR1    EVQLVESGGGLVQPGGSMRLSCAASGFTFS  Seq. ID No.: 75)CDR1   DAWMD (Seq. ID No.: 76)FR2    WVRQSPGKGLEWVA (Seq. ID No.: 77)CDR2   EIRNKANNHATYYDESVKG (Seq. ID No.: 78)FR3    RFTISRDDSKSTVYLQMNSLRAEDTALYYCTG  (Seq. ID No.: 79)CDR3   EFAN (Seq. ID No.: 80)FR4    WGQGTLVTVSS (Seq. ID No.: 81)______________________________________

__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 81(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:TGAAGCTTGCTCACTGGATGGTGGGAA27(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AGATGGGGGTGTCGTTTTGG20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 28 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GCTTGAATTCCAGGGGCCAGTGGATAGA28(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AGGTBHAHCTGCAGBAGTCWGG22(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:ATGTACTTGGGACTGAACTATGTCTT26(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:GGGGATATCCACCATGTACTTGGGACTGAACTATGTCTTCA41(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTGGT39(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GGGGCTAGCTGCAGAGACAGTGACCAGAGTCC32(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GGGGTCGACTTACGTTTGATTTCCAGCTTGGTGCCTCCA39(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 394 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGTTGTTCTGGA40TTCCTGCTTCCATCAGTGATGTTGTGATGACCCAAACTCC80ACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCTTCCATC120TCTTGCAGATCTAGTCAGAACCTTGTACACAACAATGGAA160ACACCTATTTATATTGGTTCCTGCAGAAGTCAGGCCAGTC200TCCAAAGCTCCTGATTTATAGGGCTTCCATCCGATTTTCT240GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGAGACAG280ATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCT320GGGAGTTTATTTCTGCTTTCAAGGTACACATGTTCCGTGG360ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC394(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 131 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:MetLysLeuProValArgLeuLeuValLeuLeuPheTrp1510IleProAlaSerIleSerAspValValMetThrGlnThr152025ProLeuSerLeuProValSerLeuGlyAspGlnAlaSer3035IleSerCysArgSerSerGlnAsnLeuValHisAsnAsn404550GlyAsnThrTyrLeuTyrTrpPheLeuGlnLysSerGly556065GlnSerProLysLeuLeuIleTyrArgAlaSerIleArg7075PheSerGlyValProAspArgPheSerGlySerGlySer808590GluThrAspPheThrLeuLysIleSerArgValGluAla95100GluAspLeuGlyValTyrPheCysPheGlnGlyThrHis105110115ValProTrpThrPheGlyGlyGlyThrLysLeuGluIle120125130Lys131(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 403 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:ATGTACTTGGGACTGAACTATGTCTTCATAGTTTTTCTCT40TAAAAGGTGTCCAGAGTGAAGTGAAGCTTGAGGAGTCTGG80AGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCT120TGTGCTGCTTCTGGATTCACTTTTAGTGATGCCTGGATGG160ACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGT200TGCTGAAATTAGAAACAAAGCCAATAATCATGCAACATAT240TATGATGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAG280ATGATTCCAAAAGTAGAGTGTACCTGCAAATGATAAGCTT320AAGAGCTGAAGACACTGGCCTTTATTACTGTACTGGGGAG360TTTGCTAACTGGGGCCAGGGGACTCTGGTCACTGTCTCTG400CAG403(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 134 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:MetTyrLeuGlyLeuAsnTyrValPheIleValPheLeu1510LeuLysGlyValGlnSerGluValLysLeuGluGluSer152025GlyGlyGlyLeuValGlnProGlyGlySerMetLysLeu3035SerCysAlaAlaSerGlyPheThrPheSerAspAlaTrp404550MetAspTrpValArgGlnSerProGluLysGlyLeuGlu556065TrpValAlaGluIleArgAsnLysAlaAsnAsnHisAla7075ThrTyrTyrAspGluSerValLysGlyArgPheThrIle808590SerArgAspAspSerLysSerArgValTyrLeuGlnMet95100IleSerLeuArgAlaGluAspThrGlyLeuTyrTyrCys105110115ThrGlyGluPheAlaAsnTrpGlyGlnGlyThrLeuVal120125130ThrValSerAla134(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 40 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTG40(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:ACTAGTCGACATGGAGWCAGACACACTCCTGYTATGGGT39(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 40 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:ACTAGTCGACATGGATTTWCAGGTGCAGATTTWCAGCTTC40(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CCCAAGCTTACTGGATGGTGGGAAGATGGA30(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 35 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:ACTAGTCGACATGRACTTTGGGYTCAGCTTGRTTT35(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:ACTAGTCGACATGAGAGTGCTGATTCTTTTGTG33(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:ACTAGTCGACATGGATTTTGGGCTGATTTTTTTTATTG38(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 35 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(B) LOCATION: 30(D) OTHER INFORMATION: 30 is inosine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CCCAAGCTTCCAGGGRCCARKGGATARACNGRTGG35(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 37 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG37(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:CCCGTCGACTTACGTTTTATTTCCAGCTTGGTCCCCCCT39(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GGGGATATCCACCATGGACTTTGGGCTCAGCTTGGTTTT39(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:CCCGCTAGCTGCAGAGACAGAGACCAGAGTCC32(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 394 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGA40TTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCC80TCTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATC120TCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAA160ACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTC200TCCAAAGCTCCTGATCTACAAAGTTTCCATCCGATTTTCT240GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAG280ATTTCACACTCAATATCAGCAGAGTGGAGGCTGAGGATCT320GGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTAC360ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC394(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 418 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:ATGGACTTTGGGCTCAGCTTGGTTTTCCTTGTCCTTATTT40TAAAAGGTGTCCAGTGTGAAGTGCAGATGGTGGAGTCTGG80GGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCC120TGTGCAGCCTCTGGATTCGCTTTCAGTAGCTATGCCATGT160CTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGT200CGCAGAAATTAGTAGTGGTGGTAATTACGCCTACTATCAA240GACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATG280CCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTC320TGAGGACACGGCCATGTATTACTGTGCAAGGGAGGACTAC360GGTATCCCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTC400TGGTCTCTGTCTCTGCAG418(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:MetLysLeuProValArgLeuLeuValLeuMetPheTrp1510IleProAlaSerSerSer1519(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:AspValLeuMetThrGlnThrProLeuSerLeuProVal1510SerLeuGlyAspGlnAlaSerIleSerCys152023(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:ArgSerSerGlnSerIleValHisSerAsnGlyAsnThr1510TyrLeuGlu15(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:TrpTyrLeuGlnLysProGlyGlnSerProLysLeuLeu1510IleTyr15(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:LysValSerIleArgPheSer15(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:GlyValProAspArgPheSerGlySerGlySerGlyThr1510AspPheThrLeuAsnIleSerArgValGluAlaGluAsp152025LeuGlyIleTyrTyrCys30(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:PheGlnGlySerHisValProTyrThr15(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:PheGlyGlyGlyThrLysLeuGluIleLys1510(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:MetAspPheGlyLeuSerLeuValPheLeuValLeuIle1510LeuLysGlyValGlnCys15(2) INFORMATION FOR SEQ ID NO: 37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:GluValGlnMetValGluSerGlyGlyGlyLeuValLys1510ProGlyGlySerLeuLysLeuSerCysAlaAlaSerGly152025PheAlaPheSer30(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:SerTyrAlaMetSer15(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:TrpValArgGlnSerProGluLysArgLeuGluTrpVal1510Ala14(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:GluIleSerSerGlyGlyAsnTyrAlaTyrTyrGlnAsp1510ThrValThrGly15(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:ArgPheThrIleSerArgAspAsnAlaLysAsnThrLeu1510TyrLeuGluMetSerSerLeuArgSerGluAspThrAla152025MetTyrTyrCysAlaArg30(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:GluAspTyrGlyIleProAlaTrpPheAlaTyr1510(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:TrpGlyGlnGlyThrLeuValSerValSerAla1510(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:GluValGlnMetValGluSerGlyGlyGlyLeuValLys1510ProGlyGlySerLeuLysLeuSer1520(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GluValGlnMetValGluSerGlyGlyGlyLeuValLys1510ProGlyGlyXaaLeuLysLeuSer1520(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:AspValLeuMetThrGlnThrProLeuSerLeuProVal1510SerLeuGlyAspGlnAlaSerIle1520(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:AspValLeuMetThrGlnThrProLeuSerLeuProVal1510XaaXaaGlyAspGlnAlaSerIle1520(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:AspValLeuMetThrGlnThrProLeuSerLeuProVal1510SerLeuGlyAspGlnAlaSerIle1520(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:AspValLeuMetThrGlnThrProLeuSerLeuProVal1510SerLeuGlyAspGlnAlaSerIle1520(2) INFORMATION FOR SEQ ID NO: 50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50 :TCCCTGGGTCTCACTCCTGGAGAGCCAGCTTCCATCTCTT40GCAGATCTAGT51(2) INFORMATION FOR SEQ ID NO: 51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:AGCTTGGGTCTCAGGAGTGACAGGCAGGGAGAGTGGAGAT40TGGGTCATCACAAC54(2) INFORMATION FOR SEQ ID NO: 52:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 37 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52 :GTTCCTGGTCTCGCCAGGCCAGTCTCCAAAGCTCCTG37(2) INFORMATION FOR SEQ ID NO: 53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:TTGGAGGTCTCCCTGGCTTCTGCAGGAACCAATATAAAT39(2) INFORMATION FOR SEQ ID NO: 54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:TTCACAGGTCTCATCAGCAGAGTGGAGGCTGAGGATGTGG40GAGTTTATTT50(2) INFORMATION FOR SEQ ID NO: 55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:AGCCTCGGTCTCGCTGATCTTGAGTGTGAAATCTGTCCCT40GATCCACTGC50(2) INFORMATION FOR SEQ ID NO: 56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:CCTGGAGGATCCATGAGACTCTCTTGTGCT30(2) INFORMATION FOR SEQ ID NO: 57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57 :GTTGGGGCTAGCAGAAGAGACAGTGACCAGAGT33(2) INFORMATION FOR SEQ ID NO: 58 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:TACCTGGGTCTCAATAGCTTAAGAGCTGAAGACACTGCCC40TTTATTACTGT51(2) INFORMATION FOR SEQ ID NO: 59 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:TTCAGCGGTCTCGCTATTCATTTGCAGGTACACAGTACTT40TTGGAATCATC51(2) INFORMATION FOR SEQ ID NO: 60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:GTCCGCGGTCTCCCAGGGAAGGGGCTTGAGTGGGTTGCTG40AAATTAGAAA50(2) INFORMATION FOR SEQ ID NO: 61 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 49 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61 :CTCAAGGGTCTCCCCTGGAGACTGGCGGACCCAGTCCATC40CAGGCATCA49(2) INFORMATION FOR SEQ ID NO: 62 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 52 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:TGAGGAGGTCTCAGGAGGCTTGGTGCAACCTGGAGGATCC40ATGAGACTCTCT52(2) INFORMATION FOR SEQ ID NO: 63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:GGTTGCGGTCTCCCTCCTCCAGACTCCACAAGCTGCACTT40CACTCTGGACA51(2) INFORMATION FOR SEQ ID NO: 64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 394 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGTTGTTCTGGA40TTCCTGCTTCCATCAGTGATGTTGTGATGACCCAATCTCC80ACTCTCCCTGCCTGTCACTCCTGGAGAGCCAGCTTCCATC120TCTTGCAGATCTAGTCAGAACCTTGTACACAACAATGGAA160ACACCTATTTATATTGGTTCCTGCAGAAGCCAGGCCAGTC200TCCAAAGCTCCTGATTTATAGGGCTTCCATCCGATTTTCT240GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAG280ATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGT320GGGAGTTTATTTCTGCTTTCAAGGTACACATGTTCCGTGG360ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC394(2) INFORMATION FOR SEQ ID NO: 65:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 403 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:ATGTACTTGGGACTGAACTATGTCTTCATAGTTTTTCTCT40TAAAAGGTGTCCAGAGTGAAGTGCAGCTTGTGGAGTCTGG80AGGAGGCTTGGTGCAACCTGGAGGATCCATGAGACTCTCT120TGTGCTGCTTCTGGATTCACTTTTAGTGATGCCTGGATGG160ACTGGGTCCGCCAGTCTCCAGGGAAGGGGCTTGAGTGGGT200TGCTGAAATTAGAAACAAAGCCAATAATCATGCAACATAT240TATGATGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAG280ATGATTCCAAAAGTACTGTGTACCTGCAAATGAATAGCTT320AAGAGCTGAAGACACTGCCCTTTATTACTGTACTGGGGAG360TTTGCTAACTGGGGCCAGGGGACTCTGGTCACTGTCTCTT400CTG403(2) INFORMATION FOR SEQ ID NO: 66:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:MetLysLeuProValArgLeuLeuValLeuLeuPheTrp1510IleProAlaSerIleSer15(2) INFORMATION FOR SEQ ID NO: 67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:AspValValMetThrGlnSerProLeuSerLeuProVal1510ThrProGlyGluProAlaSerIleSerCys1520(2) INFORMATION FOR SEQ ID NO: 68:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:ArgSerSerGlnAsnLeuValHisAsnAsnGlyAsnThr1510TyrLeuTyr15(2) INFORMATION FOR SEQ ID NO: 69:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:TrpPheLeuGlnLysProGlyGlnSerProLysLeuLeu1510IleTyr15(2) INFORMATION FOR SEQ ID NO: 70:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:ArgAlaSerIleArgPheSer15(2) INFORMATION FOR SEQ ID NO: 71:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:GlyValProAspArgPheSerGlySerGlySerGlyThr1510AspPheThrLeuLysIleSerArgValGluAlaGluAsp152025ValGlyValTyrPheCys30(2) INFORMATION FOR SEQ ID NO: 72:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:PheGlnGlyThrHisValProTrpThr15(2) INFORMATION FOR SEQ ID NO: 73:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:PheGlyGlyGlyThrLysLeuGluIleLys1510(2) INFORMATION FOR SEQ ID NO: 74:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:MetTyrLeuGlyLeuAsnTyrValPheIleValPheLeu1510LeuLysGlyValGlnSer15(2) INFORMATION FOR SEQ ID NO: 75:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:GluValGlnLeuValGluSerGlyGlyGlyLeuValGln1510ProGlyGlySerMetArgLeuSerCysAlaAlaSerGly152025PheThrPheSer30(2) INFORMATION FOR SEQ ID NO: 76:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:AspAlaTrpMetAsp15(2) INFORMATION FOR SEQ ID NO:77:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:TrpValArgGlnSerProGlyLysGlyLeuGluTrpVal1510Ala(2) INFORMATION FOR SEQ ID NO: 78:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:GluIleArgAsnLysAlaAsnAsnHisAlaThrTyrTyr1510AspGluSerValLysGly15(2) INFORMATION FOR SEQ ID NO: 79:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:ArgPheThrIleSerArgAspAspSerLysSerThrVal1510TyrLeuGlnMetAsnSerLeuArgAlaGluAspThrAla152025LeuTyrTyrCysThrGly30(2) INFORMATION FOR SEQ ID NO: 80:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:GluPheAlaAsn(2) INFORMATION FOR SEQ ID NO: 81:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:TrpGlyGlnGlyThrLeuValThrValSerSer1510__________________________________________________________________________

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

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Classifications
U.S. Classification536/23.53, 424/134.1, 530/387.3, 536/23.5, 424/133.1, 424/135.1
International ClassificationA61K38/00, C07K16/30
Cooperative ClassificationC07K16/3015, A61K38/00, C07K2317/34
European ClassificationC07K16/30B
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